Mendelian nightmares: the germline-restricted chromosome of songbirds

Germline-restricted chromosomes (GRCs) are accessory chromosomes that occur only in germ cells. They are eliminated from somatic cells through programmed DNA elimination during embryo development. GRCs have been observed in several unrelated animal taxa and show peculiar modes of non-Mendelian inheritance and within-individual elimination. Recent cytogenetic and phylogenomic evidence suggests that a GRC is present across the species-rich songbirds, but absent in non-passerine birds, implying that over half of all 10,500 bird species have extensive germline/soma genome differences. Here, we review recent insights gained from genomic, transcriptomic, and cytogenetic approaches with regard to the genetic content, phylogenetic distribution, and inheritance of the songbird GRC. While many questions remain unsolved in terms of GRC inheritance, elimination, and function, we discuss plausible scenarios and future directions for understanding this widespread form of programmed DNA elimination. Supplementary Information The online version contains supplementary material available at 10.1007/s10577-022-09688-3.

Introduction
Not all DNA obeys Mendel's rules of inheritance. One of the best-known cases of non-Mendelian inheritance includes cytoplasmic DNA inheritance. In most animals, the mitochondrial DNA is inherited only through females, although there are exceptions where mitochondrial DNA shows paternal or doubly uniparental inheritance (Sutherland et al. 1998;Zouros 2013;Dégletagne et al. 2021). Other more particular cases comprise the non-random segregation of B-chromosomes, sex chromosomes, centromeres, and various chromosome rearrangements via meiotic drive, which have been observed in many eukaryotes (De Villena and Sapienza 2001a, b;Camacho 2005;Rutkowska and Badyaev 2008;Yoshida and Kitano 2012;Houben 2017;Johnson Pokorná and Reifová 2021). In some organisms, sex chromosomes, sets of chromosomes, or even one of the parental genomes is eliminated during early embryogenesis, deviating from standard Mendelian inheritance (Sánchez 2014;Dedukh and Krasikova 2022). Another increasingly recognized and studied type of non-Mendelian inheritance is seen in the so-called germline-restricted chromosomes (GRCs), present in diverse multicellular organisms such as lampreys and hagfishes (reviewed by Smith et al. 2021), and some dipteran insects (reviewed by Hodson and Ross 2021). GRCs are eliminated from somatic cells through programmed DNA elimination but maintained in the germline and can show different behavior between male and female meiosis. They also sometimes show mitotic instability which results in a variable number of GRC copies in the germ cells. In this review, we critically reflect on the meiotic and mitotic behavior of the GRC of songbirds in light of all the gathered evidence since its discovery, including recent insights into its genetic content and evolution.
The first songbird GRC was described by Maria Ines Pigozzi and Alberto Solari in the zebra finch, Taeniopygia guttata (Pigozzi and Solari 1998). It was serendipitously discovered during the comparative study of the meiotic behavior of (sex) chromosomes in female and male birds. Unexpectedly, zebra finch female and male germ cells showed a large additional chromosome (in fact, the largest in the germline karyotype) that was absent in their bone marrow cells (Pigozzi and Solari 1998). This strange chromosome had not been found in other non-passerine bird species examined, i.e., pigeon, domestic chicken, and Japanese quail (Pigozzi and Solari 1999a, b;Pigozzi 2001;Calderón and Pigozzi 2006). The GRC was usually present as two copies in oocytes and as a single copy in spermatocytes (Pigozzi and Solari 2005). Another surprising feature of this GRC was its consistent elimination from the male germ cells soon before the end of meiosis, suggesting exclusive maternal transmission to the progeny (Pigozzi andSolari 1998, 2005), and thus non-Mendelian inheritance.
In its first description, the zebra finch GRC was compared to B chromosomes as they have some characteristics in common. In contrast to the essential A chromosomes, which comprise autosomes and sex chromosomes, B chromosomes are supernumerary and dispensable (Randolph 1928). B chromosomes are known to be present in only a subset of individuals within a species (or even only a subset of cells within an individual) and often accumulated via non-Mendelian mechanisms (reviewed in Johnson Pokorná and Reifová 2021). The GRC resembles a mitotically unstable B chromosome as it is present in only a subset of cells within an individual and because individuals can differ in the number of GRC they carry (Pigozzi andSolari 1998, 2005). However, it distinguishes itself by its consistent presence in the germline and obligatory absence in the soma. In fact, the GRC has been found in germ cells (except for spermatozoa) of all the individuals examined, which strongly suggests that it is indispensable for the germline and is not a standard B chromosome (Camacho 2005). Since its discovery (Pigozzi and Solari 1998), the apparent ubiquity of the songbird GRC, together with its tissue specificity, has been strongly suggestive that the GRC might be important for oogenesis and/or the early stages of spermatogenesis, but dispensable or detrimental for somatic cells. However, due to limitations in sequencing technology, the genetic content and potential function of the songbird GRC remained elusive for decades, until very recently.

Genetic content of the zebra finch GRC
The zebra finch is currently the only bird species with a sequenced germline genome (Kinsella et al. 2019). Prior to high-throughput genome/transcriptome analyses, the first and only evidence of the zebra finch GRC genomic content was the 27L4 marker identified by Itoh et al. (2009) when comparing DNA from testis and blood of the same individual using random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR). They showed that this GRC-linked sequence has homology with the short arm of the third-largest chromosome of zebra finch (i.e., chromosome 1 in the somatic reference genome; Kinsella et al. 2019), indicating for the first time that the GRC contains duplicated sequences from the regular A chromosomes. The first coding region of the GRC was only revealed 9 years later and confirmed that the GRC contains paralogous sequences duplicated from the A chromosomes; i.e., by using a subtractive transcriptomic approach, Biederman et al. (2018) characterized a GRC-linked paralog of the napa gene (napa GRC ) which showed a sequence similarity of 81% to the A-chromosomal paralog (napa A ).
Shortly thereafter, Kinsella et al. (2019) compared soma and testis genome sequencing data from three zebra finch individuals with different mitotypes, increasing the catalog to 115 high-confidence GRC-linked genes, three of which (gbe1, robo1, and dph6) were also independently detected by zebra finch GRC microdissection and sequencing (Torgasheva et al. 2019). Strikingly, each of these GRClinked genes had a paralog on the A chromosome. GRC-linked paralogs were copies of genes located on 19 different A chromosomes and showed different levels of divergence, copy number, and completeness. In contrast to protein-coding genes, the overall abundance of transposable elements and satellite DNA was lower on the GRC when compared to the A chromosomes (Kinsella et al. 2019). Both subtractive transcriptomics (Biederman et al. 2018) and analyses of coverage and single-nucleotide variants (SNVs) comparing testis and soma genome data (Kinsella et al. 2019;Pei et al. 2022) found solely GRC sequences that have an A-chromosomal paralog. These findings indicate that many, if not all GRC sequences, originated from the A chromosomes, without loss of those regions from the A chromosomes. Note that the current catalog of GRC genes is limited to high-confidence genes supported by two independent lines of evidence, SNVs between GRC and A-chromosomal paralogs, and testis/soma coverage differences. Certain GRClinked genes are likely missing from this catalog, particularly those with low sequence divergence from their A-paralog and those found in a single or low number of copies on the GRC. The latter is mainly due to the presence of somatic cells in the testes, which causes a reduction of the GRC proportion to around one GRC per every three haploid A genomes as inferred from counting testis cells stained by fluorescence in situ hybridization (FISH; Kinsella et al. 2019). Using GRC-linked SNV evidence in a linked-read testis assembly and manual curation, 36 scaffolds with a total length of 1.24 Mb were assigned to the zebra finch GRC, which represents less than 1% of its expected size (Kinsella et al. 2019). Recently, Asalone et al. (2021) developed a bioinformatic approach to reanalyze the testis assembly from Kinsella et al. (2019) and combined soma and testis re-sequencing data from four individuals from Biederman et al. (2018) and Kinsella et al. (2019). They newly identified two protein-coding genes and 733 high-confidence GRC scaffolds in the linked-read testis assembly. GRC transcription is supported by cytogenetic and transcriptomic analyses. In females, the GRC shows dispersed chromatin as found on the A chromosomes (Pigozzi and Solari 1998) and also forms the typical transcriptionally active loops associated with the phosphorylated form of the RNA pol II with transcriptionally-active chromatin during the meiotic lampbrush chromosomes stage (Torgasheva et al. 2019). However, in males, the GRC appears to be silenced in the primary spermatocytes as suggested by the labeling of the GRC with antibodies against repressive histone modifications such as H3K9me3, H3K9me2, and MacroH2A during meiotic prophase (Schoenmakers et al. 2010;del Priore and Pigozzi 2014). This evidence is consistent with qPCR data which showed higher expression of napa-GRC in the ovaries than testes (Biederman et al. 2018). Transcriptional activity was also supported by the evidence of 32 GRC-linked genes being expressed in ovaries and 6 in testes (Kinsella et al. 2019). Additionally, the same study confirmed protein expression of 5 GRClinked genes in adult gonads of both males and females. The gene composition of the GRC appears to be nonrandom, as the chromosome is enriched in gene ontologies related to embryonic and germline development (Kinsella et al. 2019). Moreover, the GRC was found to be enriched for genes that show high levels of expression in the gonads of chicken (which lack a GRC), suggesting an additional function in the ovaries and testes (Kinsella et al. 2019). These results are based on a subset of high-confidence genes and, therefore, should be interpreted with caution until there is a more complete picture of the GRC content.
The presence and expression of GRC-linked developmental genes indicate that these genes might be functional and should thus exhibit signatures of selection (Kinsella et al. 2019). Biederman et al. (2018) reported the first evidence for purifying selection, i.e., the removal of deleterious amino acid-changing SNVs, acting on a GRC-linked paralogous gene, napa GRC , by analyzing the ratio between non-synonymous and synonymous SNVs between napa GRC and napa A of zebra finch and napa A of other birds. Kinsella et al. (2019) extended this approach to all GRC-linked genes they identified with ≥ 50 SNVs between GRC and A-chromosomal paralogs (16 genes including napa) and identified 9 genes with signatures of long-term purifying selection on the GRC paralog (bicc1, cpeb1, efnb1, napa, pim1, pim3, rfc1, scrib, trim71). Interestingly, one of the GRC paralogs (puf60) was found to be under positive selection, i.e., selection to fix beneficial amino acid-changing SNVs. This gene was recently shown to exist in two haplotypes on guppy fish autosomes with opposing effects on male vs. female survival (Lin et al. 2021), suggesting a similar mechanism might have driven positive selection of the zebra finch puf60.
Judging from phylogenetic analyses, the GRClinked paralogs appear to have arrived on the GRC at different times across the evolution of Passeriformes. For instance, bicc1 GRC suggests an emergence in the oscine songbird ancestor, while trim71 GRC in the passerine ancestor. In essence, most of the GRC-linked genes emerged very recently, but some of them date back to very early in the songbird diversification, over thirty million years ago (Kinsella et al. 2019). This suggestion was further supported by a comparative cytogenetic analysis of 24 bird species (Torgasheva et al. 2019). Taken together, the GRC appears to be an amalgamation of old and new sequences. A more complete zebra finch GRC assembly and genomic data from more songbirds are needed to conclusively unravel how many genes are ancient and under selection.

Phylogenetic distribution and interspecies variation
Until recently, the only other songbird species with a reported GRC was the Bengalese finch, Lonchura striata domestica (del Priore and Pigozzi 2014). This changed when Torgasheva et al. (2019) examined pachytene chromosomes immunostained by antibodies against the synaptonemal complex (SC) of 14 songbird species and found a GRC in every one of them. They focused on the male germline because in spermatocytes, the GRC is usually present in one copy as a heterochromatic univalent heavily labeled with antibodies against centromere proteins and therefore is easily distinguishable from the bivalents formed by the regular A-chromosomes (Fig. 1a). In females, the GRC is usually present in two copies forming a recombining bivalent (Fig. 1b). Torgasheva et al. (2019) also showed the absence of the GRC in another 8 non-passerine species from seven different lineages by re-analyzing previously published images of spermatocytes spreads. Recently, the GRC was found in 11 additional songbird species (Slobodchikova et al. 2022;Poignet et al. 2021;Sotelo-Muñoz et al. 2022).
The presence of a GRC in every songbird species cytogenetically analyzed so far (Fig. 2) suggests a monophyletic origin of the GRC at least in the common ancestor of the sampled songbirds (i.e., spanning the vast majority of Oscines). This hypothesis is supported by the phylogenetic analysis of bicc1 GRC from the zebra finch relative to bicc1 A across birds (Kinsella et al. 2019). Although deep-branching Oscines, Suboscines, and Acanthisitti species have not been examined yet, they probably contain a GRC as well, because the zebra finch trim71 GRC branches outside of trim71 A from all passerines, suggesting emergence of the GRC paralog in the common ancestor of passerines (Kinsella et al. 2019). Taken together, this indicates that the GRC is present in at least all songbirds, the largest group of birds comprising half of all bird species, and at most in all passerines, nearly two thirds of all bird species (Oliveros et al. 2019).
Cytogenetic analysis revealed that the GRCs show remarkable interspecies variation in size. Torgasheva et al. (2019) classified them broadly as macro-and micro-GRCs (i.e., either belonging to the macro-or microchromosome set of the species). Macro-GRCs were found in 12 species, micro-GRCs in 14 species, and one species showed a mosaic individual containing both macro-and micro-GRCs (Slobodchikova et al. 2022;Poignet et al. 2021;Sotelo-Muñoz et al. 2022). Furthermore, there is no phylogenetic Fig. 1 Pachytene spermatocytes of the pale martin (a, d) and oocytes of the sand martin (b, c) after immunostaining with antibodies against the synaptonemal complex: SYCP3 (red), centromere proteins (blue), and recombination foci: MLH1 (green). The GRC is usually present as one copy forming a univalent in primary spermatocytes (a) and as two copies forming a recombining bivalent in primary oocytes (b).
Some females contain only one copy forming a univalent in all oocytes (polymorphism) (c). Some males contain two partially synapsed GRC copies in few spermatocytes (mosaicism) (d). clustering according to GRC size (Fig. 2), suggesting multiple events of massive additions, deletions, and amplifications occurring on the GRC, sometimes even among very closely related species or even within species, such as in the black-headed munia (Sotelo-Muñoz et al. 2022).
In order to estimate divergence of genetic content between GRCs of different species, Torgasheva et al.
(2019) prepared whole-GRC microdissected DNA probes. The results of reciprocal FISH with these probes demonstrated substantial genetic divergence between GRCs of different species. The intensity and coverage of the hybridization signal on the GRC decreased when the phylogenetic distance between the species from which the probe was derived and the target species increased (Torgasheva et al. 2019(Torgasheva et al. , 2021. Whole-GRC probes derived from different species hybridized with different regions of the A chromosomes. For example, the zebra finch GRC probe labeled the region on the short arm of the thirdlargest chromosome (i.e., homologous to zebra finch chromosome 1) in all species examined (Torgasheva et al. 2019), while the great tit GRC probe labeled a part of the W chromosome (Torgasheva et al. 2021). These findings suggest that GRCs of different species contain different multiply repeated regions homologous to regions on the A chromosomes.

The mystery of GRC inheritance
Despite all the information gathered in the almost 25 years since its discovery, it is still not known how the GRC is passed down through the generations. One reason for this is that even though there is a lot of information about the behavior of the GRC in spermatogenesis and in the early stages of female meiosis, knowledge about its behavior during early embryo development and late stages of female gametogenesis is still lacking. In an attempt to explain the differences in GRC copy number between sexes (as well as the occasional variation in GRC copy number in the same sex or even within a single individual), possible scenarios of GRC inheritance have been proposed that will be discussed below.

Maternal inheritance of a single GRC copy
Cytogenetic studies of the GRC have predominantly focused on examining the GRC during meiosis (Fig. 3a). In males, the GRC is normally observed as a single univalent chromosome in spermatogonia (Pigozzi and Solari 1998;del Priore and Pigozzi 2014) and primary spermatocytes (Pigozzi and Solari 2005;Torgasheva et al. 2019). This single GRC is eliminated from the nucleus during the first meiotic division and forms a micronucleus that is later ejected from the cell (Pigozzi andSolari 1998, 2005;del Priori and Pigozzi 2014). In females, the GRC is usually found as two copies (i.e., a paired bivalent, resembling autosomes) that engage in regular crossing-over (Fig. 1b;Pigozzi and Solari 2005;del Priori and Pigozzi 2014). These observations led to the view that the GRC is only inherited from the maternal side (Fig. 3a). A case of maternal inheritance of the GRC was recently reported in F1 hybrids between two different munia species (Lonchura spp.) with distinct GRC size, where the F1 hybrids' GRC matched the size of the maternal species' GRC (Sotelo-Muñoz et al. 2022). In addition, sequencing of germline samples (testis and ejaculates) from multiple male zebra finches from the same family showed that all brothers shared the same GRC haplotype as the brother of their mother (Pei et al. 2022). However, the apparent uniparental inheritance of a single GRC copy raises the question about when and how a second copy arises in the female, but not in the male. Pigozzi and Solari (2005) suggested that the sex difference in GRC copy number arises during the germline/soma differentiation. They proposed that in females, nondisjunction of the GRC sister chromatids during a mitotic division may produce a germline progenitor cell with two GRC copies and a somatic progenitor cell with no GRC. In males, one GRC chromatid may be transmitted to the germline progenitor cell and the other one lost (presumably by chromatid lagging during mitotic anaphase), leading to a somatic progenitor cell without a GRC, and  Poignet et al. (2021). The phylogenetic tree is a dated supertree combining Jarvis et al. (2014) for deep avian branches, Oliveros et al. (2019) for deep songbird (Oscines) branches, and TimeTree.org consensus estimates for the remaining more recent branches (Kumar et al. 2017). Note that divergence estimates for deep avian/oscine relationships vary significantly between studies (reviewed by Suh 2016), e.g., the divergence of Estrildidae and Corvidae (common ancestor of the sampled Oscines here) was estimated as 30.6 mya by Oliveros et al. (2019) and 52.9 mya by Ericson et al. (2014). The present supertree shows lower ends of such estimates as a conservative minimum age for GRC emergence, and we proportionally scaled the TimeTree.org branch length estimates within Fringillidae, Muscicapidae, Hirundinidae, and Galliformes relative to each respective outgroup ◂ a germline with a single GRC (Fig. S1). Pigozzi and Solari (2005) hypothesized that sex-biased expression of cohesin-related genes might be involved in these differences in chromosome behavior. This explanation provides a mechanism to explain not only sex differences in GRC copy number but also how the GRC is lost from somatic cells.
However, it is also possible that the sex-specific difference in GRC copy number does not arise during the differentiation between germline and soma (Fig. 3b), but at a later stage, for example, during mitotic divisions of primordial germ cells (PGCs; Fig. 3c). Nondisjunction of GRC sister chromatids during the mitosis of female PGCs might be a simple mechanism that could lead to one cell without the GRC (which might undergo apoptosis or become a somatic cell) and another cell with two GRC copies that are able to synapse and recombine during meiosis. Such an asymmetrical cell division with GRC non-disjunction would need to be strictly regulated to occur only once and only in females.

Occasional paternal inheritance of the GRC
While maternal inheritance appears to be the norm, very recent studies suggest that the GRC can occasionally be paternally inherited. Pei et al. (2022) demonstrated that the GRC can occasionally be paternally inherited based on three main findings: (1) a hybrid individual between the two zebra finch subspecies a In males, the GRC is typically observed as a single copy in spermatogonia which is eliminated later during spermatogenesis. In female nestlings, the GRC is normally observed as two copies in the primary oocytes that go through regular meiotic recombination. b According to Pigozzi and Solari (2005), both male and female zygotes maternally inherit a single copy of GRC. During early mitotic divisions, elimination of the GRC in some cells might result in somatic cells whereas cells with regular mitosis would result in primordial germ cells. c After germline/soma differentiation, the single-copy GRC in male primordial germ cell follows regular mitotic behavior. In young females, the GRC might show nondisjunction of sister chromatids during a pre-meiotic cell division, generating one daughter cell containing no GRC and a primary oocyte with two copies of the GRC. Some females or cells may present normal mitosis (i.e., without GRC non-disjunction) resulting in females or cells containing a single-copy of the GRC (polymorphism or mosaicism in females). Also, note that "duplication" of the GRC may occasionally occur during male mitotic division, and this may explain the observation of males with 2-3 copies of the GRC in some of their germ cells (mosaicism in males) exhibiting mitochondrial DNA from the maternal subspecies but a GRC from the paternal subspecies, (2) a striking topological incongruence between mtDNA and GRC haplotype trees, suggesting that at least some GRC haplotypes were able to cross matriline boundaries, and (3) the presence of the GRC in a small portion of sperm heads. Interestingly, Pei et al. (2022) found that males varied substantially in the proportion of spermatozoa (1-19%) that contained the GRC, and that this pattern is family-specific, with males from the same family showing a consistently low or high proportion of GRC retention in their spermatozoa. This suggests a heritable component for GRC presence in sperm cells. The GRC was also observed in spermatozoa of great tits, although it was rare (in 3 out of 880 spermatozoa; Torgasheva et al. 2021).
It is currently unclear what happens when the zygote receives two copies of the GRC, one of maternal and one of paternal origin. Pei et al. (2022) did not observe any GRC-heterozygous individuals in their sample, but this may simply be a consequence of biparental inheritance being uncommon. If receiving two copies of the GRC would cause substantial problems during embryonic development, such paternal inheritance should be selected against. However, if no problems arise, the strategy of biparental inheritance should rapidly outcompete the strategy of restriction to maternal inheritance, simply because GRCs that are biparentally inherited are more likely to be passed on to the next generation. The observed polymorphism and high repeatability in the effectiveness of GRC elimination during spermatogenesis open up the possibility to study and evaluate the success of the paternal inheritance strategy in the future.

Polymorphism and mosaicism in GRC copy number and size
The GRC is usually present as two copies in oocytes and as a single copy in spermatocytes (Pigozzi and Solari 2005). However, polymorphism (i.e., variation among individuals) and mosaicism (i.e., variation within the same individual) in GRC copy number have been observed in males and females of some species (Table 1). In the zebra finch (Pigozzi andSolari 1998, 2005) and the sand martin Riparia riparia ( Fig. 1c; Malinovskaya et al. 2020a), female individuals with a single GRC copy in all their primary oocytes have been found, although in a relatively low proportion (12% of zebra finch females and 17% of sand martin females; Table 1). In the great tit Parus major, four of seven females showed mosaicism for GRC copy number, with the majority of primary oocytes containing two GRC copies and the minority (from 2 to 26%) a single copy (Torgasheva et al. 2021). These observations might be explained by the failure of GRC duplication in some or all PGCs during female embryonic development (Fig. 3c). It is plausible that GRC duplication might not be absolutely essential, assuming that in females with a single GRC copy, the unpaired GRC univalent might remain in the egg cell while the polar body does not receive any GRC. The non-negligible frequency in which this has been observed in zebra finch and sand martin populations might mean that females with a single GRC copy do not have a dramatically reduced fitness (compared to females with two GRC copies) and that the duplication of the GRC in females is not under strong selection pressure.
In males, mosaicism in GRC copy number has been observed in four species (Table 1). In pale martins, Riparia diluta, seven out of nine analyzed males showed GRC copy number mosaicism in primary spermatocytes ( Fig. 1d; Malinovskaya et al. 2020a). In these males, most primary spermatocytes had a single GRC copy, but spermatocytes with two or even three copies were also observed. Similar observations were described in the great tit (Torgasheva et al. 2021) and black-headed munia (Sotelo-Muñoz et al. 2022), wherein some spermatocytes with two GRC copies were observed. In one of the two analyzed individuals of European pied flycatcher, most of the primary spermatocytes surprisingly carried two GRCs (Slobodchikova et al. 2022). The relatively high number of species in which such mosaicism has been observed could suggest that the segregation of the GRC during mitosis is often unstable in males. Occasional non-disjunction of GRC sister chromatids during mitotic divisions of PGCs might be the reason why some spermatocytes carry more than a single GRC copy, as it has been observed for B chromosomes in some species (Nur 1963;Jones 2018). It is plausible that such variability may be inconsequential since all GRC copies seem to be canonically eliminated during spermatogenesis (Pigozzi and Solari 2005;   Polymorphism and mosaicism were observed not only for GRC copy number but also for GRC size. In one male black-headed munia, a small proportion of spermatocytes contained two GRCs, either a micro-GRC and a macro-GRC or else two micro-GRCs (Sotelo-Muñoz et al. 2022). This suggests that significant variation in the GRC size may exist not only between species but also within species and even within a single individual. Sotelo-Muñoz et al. (2022) suggested several mechanisms which could explain the origin of within-species polymorphism in GRC size. For example, fragmentation of the GRC during its elimination from the spermatocytes followed by paternal inheritance of the GRC fragment can lead to the origin of a smaller GRC in a population. A shorter GRC might also be the result of GRC fragmentation and loss of its parts during germline mitotic divisions. This sort of mutation would normally not be tolerated by the cell in standard A chromosomes, but given the enormous variability in GRC size even among closely related species, it is possible that large parts of this chromosome are in fact non-essential and thus their loss might not have large effects on their carrier's fitness. At the same time, additions of new sequences to the GRC from standard chromosomes may be well tolerated as the presence of the GRC only in the germline reduces the pleiotropic effects of such mutations. It is plausible that once polymorphism in the GRC size exists in the population, occasional inheritance of two GRCs of different sizes by a single zygote and unstable mitotic inheritance of these GRCs may result in the observed GRC size mosaicism.
Currently, the frequency of polymorphism and mosaicism for GRC copy number and size in songbirds is difficult to estimate. Most species have only had a few individuals analyzed, and most of these individuals were males, making estimates for females especially uncertain. However, the data obtained to date indicates that polymorphism and mosaicism in GRC copy number could be relatively frequent across songbird species (Table 1).

Female meiotic drive and maternal inheritance of two GRC copies
An alternative explanation for where the two GRCs in females come from, as well as why polymorphism and mosaicism for GRC number occur, was proposed by Malinovskaya et al. (2020a). They suggested that zygotes of both males and females can already contain two GRC copies. Both copies would be inherited from the mother due to nondisjunction of GRC homologs in the first meiotic division (MI) and their preferential segregation into the egg (i.e., meiotic drive). During germline development, germ cells can actively eject or passively lose one of the GRCs. Since male germ cells undergo a much higher number of mitotic divisions before entering meiosis, they would be more likely to lose one of the GRCs and contain a single copy in the pachytene cells. This scenario does not exclude the possibility that zygotes with one GRC copy occasionally arise via normal segregation of two GRCs in female meiosis I. A single GRC could also be inherited from mothers carrying a single GRC in their pachytene cells, which would explain why some females have only one GRC.
This scenario could potentially explain sex differences, polymorphism, and mosaicism in GRC copy * Number of mosaic birds and the percentage of cells that differed from the norm for that sex (i.e., 2 for female and 1 for male). **The frequencies of cells carrying more than 1 GRC were estimated from both premeiotic and meiotic cells. One of the Riparia diluta male also had 3 GRCs in 6% of pachytene cells. ***In addition to 5% differing in GRC number, cells also differed in GRC size with 37% micro-GRC and 58% macro-GRC. †References: 1: Pigozzi and Solari (1998); 2: Pigozzi and Solari (2005)  number. However, it depends on the validity of its key assumption: meiotic drive via nondisjunction and preferential segregation of both GRC homologs into the secondary oocyte (and then to the egg cell after normal segregation in meiosis II). In birds, the only known asymmetric divisions, which could provide a high efficiency of GRC accumulation, occur during female meiosis. Both polar bodies are formed at the periphery of the oocyte; therefore, if GRC homologs do not separate, they have a high chance to remain in the egg. Indeed, meiotic drive of B chromosomes during asymmetrical MI has been documented in females of many non-avian species (Hewitt 1976;Nur 1977;Nur and Brett 1985;Cano and Santos 1989;Santos et al. 1993). However, these studies mostly described the drive of a single B chromosome, which formed a univalent. Meiotic drive of GRC in MI requires nondisjunction of properly synapsed GRC bivalents. Malinovskaya et al. (2020a) suggested that nondisjunction can be facilitated by the extreme polarization of chiasmata positions in GRC bivalents. In females of all three species carrying macro-GRCs studied to date (zebra finch, sand martin, and great tit), recombination occurs in one or both ends of GRC bivalents ( Fig. 1b; Pigozzi and Solari 2005;Malinovskaya et al. 2020a;Torgasheva et al. 2021). Such a polarized distribution of chiasmata is associated with an increased frequency of chromosome nondisjunction at the first meiotic division in other organisms (Sears et al. 1995;Koehler et al. 1996;Hassold and Hunt 2001). However, recent observations reporting the lack of heterozygosity in zebra finch male siblings, which share the same GRC haplotype as their uncle from their maternal side (Pei et al. 2022), contradict the assumption that two homologous GRCs are transmitted to the progeny. They must have accumulated noticeable differences if passed through many generations and recombined in limited regions. Nevertheless, female meiotic drive at MI and inheritance of two GRCs from females could possibly occur at least in some species or individuals.
Zygotes with two GRCs can also occur via nondisjunction of GRC sister chromatids and their preferential segregation to the egg cell in the second meiotic division (MII; Fig. 4). Meiotic drive in MII, although less intuitive than in MI, can also occur due to the asymmetrical geometry of this division (reviewed in Clark and Akera 2021). One may speculate that meiosis-specific cohesins or other meiotic players controlling the correct separation of sister chromatids or centromeres might be involved in the GRC nondisjunction at MII in a similar way as has been described for B chromosome drive during the first pollen mitotic division (Ruban et al. 2020). Occasional normal disjunction in MII and rare nondisjunction during premeiotic mitoses can explain how polymorphism and mosaicism for GRC copy number arise in females. In addition, under this scenario even females with a single GRC would produce gametes with two GRC copies, thereby maintaining the polymorphism in GRC copy number in the population.

GRC elimination from somatic cells and male germ cells
Another question is how the GRC is eliminated from somatic cells during early embryogenesis and from male germ cells in spermatogenesis. Currently, the mechanisms of GRC elimination from somatic cells remain entirely unknown. Pigozzi and Solari (2005) hypothesized that GRC elimination from somatic cells occurs via different mechanisms in males and females. In females, nondisjunction of the GRC chromatids and their segregation to a germline progenitor cell would leave a somatic progenitor cell without a GRC. In males, lagging of one of the GRC chromatids during mitotic anaphase would lead to a somatic progenitor cell without a GRC and a germline progenitor cell with a single GRC (Fig. S1). Such sexspecific differences in GRC behavior would, however, require sex differences in gene expression already at early stages of embryo development when the germline is determined. Alternatively, the GRC might Fig. 4 Hypothetical scenario of GRC transmission via meiotic drive in females and programmed elimination in males. Gray shading indicates the hypothetical events without cytogenetic support. According to the scenario, both males and females normally inherit two GRC copies from the mother. Both GRC copies are passively lost or actively eliminated in somatic cell lineages of both sexes. In male germline, one of the GRC copies is eliminated during early pre-meiotic mitotic divisions, the other during meiotic divisions. Delayed elimination of the first GRC copy may lead to mosaicism in males. In female germline, two GRC copies follow regular mitotic behavior. They form a bivalent and recombine in the meiotic prophase, properly segregate in the first meiotic division, non-disjoin and preferentially segregate to the egg cell in the second meiotic division (MII). Some females may present proper segregation in MII resulting in zygotes with single GRC, which can explain polymorphism in females ◂ be epigenetically modified in both sexes and marked for elimination from somatic cells in a similar way as it has been observed in spermatogenesis (del Priore and Pigozzi 2014;Malinovskaya et al. 2020a). Cytological observations of the earliest stages of songbird embryonic development are needed to shed light on the details of the GRC elimination from somatic cells.
A few pilot studies on mechanisms of GRC elimination from male germ cells during spermatogenesis have already been published (del Priore and Pigozzi 2014; Goday and Pigozzi 2010;Malinovskaya et al. 2020a;Schoenmakers et al. 2010). They showed that from the very beginning of meiotic prophase, the single GRC is heterochromatic in primary spermatocytes, marked with specific histone modifications during prophase (e.g., H3K9me3, H3K9me2, and MacroH2A), and shifted to the nuclear periphery. The GRC is then observed in the cytoplasm of secondary spermatocytes, suggesting that its elimination from the nucleus occurs during the first meiotic division. Later, the GRC is seen as a micronucleus in the cytoplasm of secondary spermatocytes and young spermatids, and finally expelled from the cells. A similar mechanism might be involved in GRC elimination from male germ cells during their pre-meiotic mitotic division if a zygote receives two GRC copies (see above). This is supported by cytological observations of pale martin spermatogonia containing two GRC copies, one of which is located within the nucleus and the other one is moved to the cell periphery and almost expelled (Fig. 5c in Malinovskaya et al. 2020a).

Evolutionary significance
The likely presence of the GRC in > 5,000 songbird species with mostly two copies in females and one copy in males, and the signatures of long-term purifying or positive selection on some zebra finch GRC-linked genes make it very tempting to speculate about the evolutionary significance of GRCs. As our conservative minimum estimate of GRC emergence is 30 mya in the ancestor of songbirds (Fig. 2), we emphasize that such an ancient origin of the songbird GRC may lead to the difficulty of distinguishing between the (potentially different) reasons that the GRC might have originally evolved for vs. what it might be doing now. Kinsella et al. (2019) defined 115 high-confidence genes out of a total of 267 candidate genes located on the zebra finch GRC, suggesting that the GRC is a gene-rich chromosome with an amalgam of paralogs from across the A chromosomes. While the remaining candidate genes await further verification, the 115 high-confidence genes alone provide ample opportunity to deliberately pick genes that point in different speculative directions: i. What if the GRC is simply a very successful parasitic B chromosome? B chromosomes are supernumerary dispensable chromosomes, which occur in a single or multiple copies in the cell and often show unstable meiotic and mitotic inheritance, which may result in their loss from the population. Because they usually do not provide any advantage to their carriers (but see Johnson Pokorná and Reifová 2021 for exceptions) and can be even harmful especially if they occur in odd copy numbers (Camacho et al. 2004), many B chromosomes evolved mechanisms to increase probability of their inheritance in a selfish way (Jones 2018). Some B chromosomes show meiotic drive increasing the chance of their transmission to the germ cells, while others, for example, show mitotic drive leading to the preferential segregation of both B chromosome chromatids to the germline (gonotaxis). Similarities between B chromosomes and GRCs have been previously noted (Johnson Pokorná and Reifová 2021) and it seems plausible that the latter might be a way of long-term stabilization of a B chromosome, i.e., a GRC would be a B chromosome with stable germline inheritance and somatic elimination. Such a B-chromosomal origin would be consistent with the female bias in GRC inheritance, akin to meiotic drive of B chromosomes often through the asymmetry of female meiosis (Jones 2018;Clark and Kocher 2019). The songbird GRC may represent such an extraordinarily successful B chromosome, which found a way to prevent its loss, albeit retaining many B chromosome characteristics including unstable mitotic inheritance and possibly female meiotic drive. In line with this, it is worth noting that the zebra finch GRC contains paralogs of cenpj (Kinsella et al. 2019), a gene involved in centriole architecture (Hatzopoulos et al. 2013) and thus compatible with speculation about the GRC ensuring its own transmission. In terms of chromosome organization, a recent high-quality assembly of the B chromosome of the Mexican cavefish Astyanax mexicanus bears striking resemblance to the GRC as it also contains high-copy paralogs from across the A chromosomes (Imarazene et al. 2021). ii. What if the GRC is involved in sexual conflict (resolution) or is even a sex chromosome? Stöck et al. (2021) recently noted that the usual songbird situation of one GRC in males and two GRCs in females resembles a germline-limited X0/XX sex chromosome system, i.e., on top of the organism-wide ZZ/ZW sex chromosome system of birds. For the at least six Z-paralogous genes on the GRC (Kinsella et al. 2019), this would effectively balance the dosage of these genes between males and females given the lack of global dosage compensation in birds (reviewed in Stöck et al. 2021). Considering that GRC inheritance might not be strictly maternal leaves additional room for sexual conflict or resolution thereof (Pei et al. 2022), and the presence of the puf60 gene on the zebra finch GRC with signatures of positive selection (Kinsella et al. 2019) brings to mind recent evidence from guppies where different alleles of this A-chromosomal gene link to male vs. female survival in the wild (Lin et al. 2021). iii. What if the GRC is involved in germline development or even a germline determinant? Kinsella et al. (2019) noted that several GRClinked genes, including the so far oldest genes trim71 GRC and bicc1 GRC , are involved in cell differentiation and germline development. trim71 GRC and bicc1 GRC have been under longterm purifying selection, bicc1 GRC mRNA was found expressed in adult ovary, and the 115 high-confidence GRC-linked genes are enriched for genes involved in female gonad development (Kinsella et al. 2019). While it remains unclear whether the GRC is expressed during early embryo development as GRC expression analyses have been limited to adult male and female gonads, these patterns would be in line with the explanation for programmed DNA elimination invoked for lampreys: The germline limitation of specific genes (or specific paralogs) might allow the evolution of germline-beneficial functions without detrimental effects when misexpressed in the soma (Smith et al. 2012;Smith 2017), i.e., minimizing antagonistic pleiotropy.
In line with this hypothesis, GRCs might be an irreversible means of avoiding germline gene misexpression in the soma, with the potential to act as a germline determinant (Kinsella et al. 2019). Germline gene misexpression in species without programmed DNA elimination (e.g., humans) has led to the notion of "cancer/testis antigens" as a form of antagonistic pleiotropy through oncogenesis in the soma (Simpson et al. 2005;Sandhu et al. 2021).
However, what if the GRC is none of these or rather several of these? Until there is a full understanding of GRC gene content across and within species, we recommend that speculation based on cherry-picked genes, no matter how tempting, should be taken with a grain of salt.

Conclusion and future directions
Although the songbird GRC has been known for nearly 25 years, only high-throughput multi-omics and comparative cytogenetic studies during the last 5 years have elevated GRCs from a niche oddity of zebra finches to a general phenomenon of all songbirds if not all passerines, i.e., at least half and up to two thirds of all 10,500 bird species. While many mysteries remain, especially with regards to when and how the GRC is transmitted or eliminated, we are positive that the newly developed genomics and cytogenetic approaches will allow high-resolution tracing of GRC presence or absence across embryogenesis and gametogenesis of zebra finches over the next years. To understand the evolutionary significance and potential adaptive value of the GRC, characterizing within-species and between-species GRC genetic diversity and gene content is needed. Seeing that GRCs of thousands of passerine species await cytogenetic and genomic characterization, the songbird GRC serves as an important reminder that multitudes of GRCs or other germline/soma genome differences are waiting to be discovered across the tree of life.


Characterization of the O-Glycoproteome of Porphyromonas gingivalis

ABSTRACT Porphyromonas gingivalis is an important human pathogen and also a model organism for the Bacteroidetes phylum. O-glycosylation has been reported in this phylum with findings that include the O-glycosylation motif, the structure of the O-glycans in a few species, and an extensive O-glycoproteome analysis in Tannerella forsythia. However, O-glycosylation has not yet been confirmed in P. gingivalis. We therefore used glycoproteomics approaches including partial deglycosylation with trifluoromethanesulfonic acid as well as both HILIC and FAIMS based glycopeptide enrichment strategies leading to the identification of 257 putative glycosylation sites in 145 glycoproteins. The sequence of the major O-glycan was elucidated to be HexNAc-HexNAc(P-Gro-[Ac]0-2)-dHex-Hex-HexA-Hex(dHex). Western blot analyses of mutants lacking the glycosyltransferases PGN_1134 and PGN_1135 demonstrated their involvement in the biosynthesis of the glycan while mass spectrometry analysis of the truncated O-glycans suggested that PGN_1134 and PGN_1135 transfer the two HexNAc sugars. Interestingly, a strong bias against the O-glycosylation of abundant proteins exposed to the cell surface such as abundant T9SS cargo proteins, surface lipoproteins, and outer membrane β-barrel proteins was observed. In contrast, the great majority of proteins associated with the inner membrane or periplasm were glycosylated irrespective of their abundance. The P. gingivalis O-glycosylation system may therefore function to establish the desired physicochemical properties of the periplasm. IMPORTANCE Porphyromonas gingivalis is an oral pathogen primarily associated with severe periodontal disease and further associated with rheumatoid arthritis, dementia, cardiovascular disease, and certain cancers. Protein glycosylation can be important for a variety of reasons including protein function, solubility, protease resistance, and thermodynamic stability. This study has for the first time demonstrated the presence of O-linked glycosylation in this organism by determining the basic structure of the O-glycans and identifying 257 glycosylation sites in 145 proteins. It was found that most proteins exposed to the periplasm were O-glycosylated; however, the abundant surface exposed proteins were not. The O-glycans consisted of seven monosaccharides and a glycerol phosphate with 0–2 acetyl groups. These glycans are likely to have a stabilizing role to the proteins that bear them and must be taken into account when the proteins are produced in heterologous organisms.

Glycosylation of P. gingivalis proteins has been reported in many studies and likely results from multiple glycosylation pathways (11). The most studied is the type IX secretion system (T9SS) sortase pathway, which brings together the synthesis of glycans associated with A-lipopolysaccharide (A-LPS) and its conjugation to T9SS cargo proteins via the PorU sortase (12,13). The sortase is a transpeptidase that cleaves the C-terminal signal domain and creates a new peptide bond between the C-terminus of the matured cargo protein and the seryl component of the linking sugar (13). The linking sugar was determined to be 2-N-seryl, 3-N-acetylglucuronamide, which is assumed to be part of the A-LPS molecule (14). The lipid portion of A-LPS is then thought to be the membrane anchor that allows the cargo proteins to be tethered to the cell surface. Biosynthesis of the linking sugar was shown to involve both the Wbp pathway as well as VimA and VimE (14,15). The glycosyltransferases shown to be specifically involved in the synthesis of A-LPS and cargo modification are WbaP, VimF, GtfC and GtfF (16). The effect of "A-LPS" modification of T9SS cargo proteins can be seen from their migration on SDS-PAGE as diffuse bands spread over a broad molecular weight (MW) typically 20-40 kDa higher than predicted from the protein sequence alone (17)(18)(19)(20).
The most important cargo proteins are the gingipains, RgpA, RgpB, and Kgp, due to their major roles in nutrient acquisition and virulence (21). In addition to the C-terminal modification with A-LPS, the RgpA catalytic domain has also been reported to be glycosylated with a smaller glycan (22), indicating the possible presence of additional systems. Other proteins that appear to be glycosylated include BamA (PG0191), whose glycosylation was dependent on the GalE putative epimerase (23); the minor fimbrillin, Mfa1 (24) based mainly on carbohydrate staining; and PGN_0743, PGN_0876, PGN_1513, and PGN_0729 (Omp41), which were also identified based on carbohydrate staining (25). Omp41 together with its binding partner Omp40 could also be isolated by lectin-affinity purification, and a putative O-glycosylation site in Omp41 was identified at Ser 271 in the sequence 269 PVSCPECPEVTPVTK 283 involving a single hexose (26). While these studies have hinted at the presence of glycosylation in P. gingivalis, others have highlighted that the conserved enzymes and antibody cross-reactivity to glycans produced in other members of the Bacteroidetes phylum support that P. gingivalis possesses a functional Bacteroidetes phylum O-glycosylation system (27,28).
The Bacteroidetes phylum O-glycosylation system was first described in Bacteroides fragilis (29). The glycosylation site motif in B. fragilis was elegantly demonstrated by sitedirected mutagenesis to be (D)(S/T)(A/I/L/V/M/T) with the glycan O-linked to the Ser or Thr residue in the second position of the motif (29). The glycosylation was shown to only occur on proteins exported beyond the cytoplasm. Furthermore, deletion of an essential component of the glycosylation machinery resulted in impaired colonization of mouse intestines, demonstrating the in vivo importance of this glycosylation system (29). Over 1,000 glycoproteins are predicted in B. fragilis based on the presence of the O-glycosylation motif, and a total of 20 glycoproteins were identified (30).
Similarly, the O-glycosylation system of the closely related Tannerella forsythia has been reported with the identification of 13 glycopeptides from 4 glycoproteins with inferred glycosylation sites consistent with the motif found in B. fragilis (28). The structure of the glycan in T. forsythia ATCC 43037 was solved by NMR and MS methods to contain 10 sugars (28,31). While both the O-oligosaccharyltransferase responsible for transferring the glycan to the protein as well as the glycosyltransferases involved in the biosynthesis of the "core" region of the glycan are unknown, the biosynthetic pathway of the "species-specific" portion of the glycan has been elucidated (31). The biosynthesis of the glycan, its variation between strains, and its roles in virulence were recently reviewed (32). Also recently, we completed a proteomics study of T. forsythia and identified 312 O-glycosylation sites in 145 glycoproteins and extended the glycosylation site motif to (D)(S/T)(A/I/L/V/M/T/S/G/C/F) (33).
In the present study, we demonstrate the presence of an O-glycosylation system in P. gingivalis for the first time with the mass spectrometry identification of 257 putative O-glycosylation sites within 145 glycoproteins together with the sequence of the glycans and the identification of two glycosyltransferases required for the glycan synthesis.

RESULTS
Overall identification data. P. gingivalis was fractionated, and samples with or without partial deglycosylation were digested with trypsin and analyzed by LC-MS/MS as outlined (Fig. 1). These analyses described in detail below ultimately led to the identification of 257 O-glycosylation sites within 145 glycoproteins (Tables S2 and S3 in the  supplemental material).
Survey of glycoforms. To investigate the anticipated O-glycoproteome of P. gingivalis, a total membrane sample was prepared, digested with trypsin, and analyzed by LC-MS/MS with two FAIMS settings and multiple fragmentation modes. The data were searched with Byonic using a wildcard setting to enable modified peptides of any Dmass to be identified. A plot showing the number of identified peptides for frequently observed Dmass values exhibited several clusters of potential glycopeptides differing by 1 Da units (Fig. 2). The most frequent Dmass values observed were the 1436 Da and 1394 Da clusters representing 242 peptides (5.3%) (Fig. 2). Other abundant values were 128.10 and 156.10, which could be attributed to Lys and Arg respectively, presumably FIG 1 Overview of sample preparation. P. gingivalis cells were divided into membrane and soluble fractions. A portion of the membrane fraction was digested with trypsin and the resultant peptides were analyzed by LC-MS/MS with or without HILIC enrichment. A second portion of the membrane sample was extracted with detergent, subjected to SDS-PAGE, and divided into 8 gel segments for digestion with trypsin (indicated by scissors) and LC-MS/MS. The soluble fraction as well as a third portion of the membrane sample underwent partial deglycosylation in trifluoromethanesulfonic acid (indicated by the hazard symbol) followed by SDS-PAGE separation into 16 gel segments, trypsin digestion and LC-MS/MS. due to the misassignment of peptides with two adjacent tryptic cleavage sites. Additional clusters were observed with Dmass values around 792 Da and 1352 Da.
A preliminary look at the MS/MS spectra of the identified peptides with potential modifications of ;1352, ;1394, and ;1436 Da indicated they were glycopeptides due to the presence of sugar fragmentation and the HexNAc oxonium ion at m/z 204.086 (see below). Furthermore, nearly all of the peptides contained the putative Bacteroidetes O-glycosylation motif of (D)(S/T)(X) (see below), suggesting that these modifications may be the major O-glycans of P. gingivalis. Peptides of the 792-cluster tended to have the same O-glycosylation motif but lacked the HexNAc oxonium ion. The glycosylated proteins appeared to be mostly lipoproteins and proteins associated with the inner membrane (IM). Therefore, to enrich for these proteins and allow a deeper survey of the Oglycoproteome, the total membrane sample was extracted with a low concentration of detergent and fractionated by SDS-PAGE (Fig. 1). Trypsin digestion of the gel segments followed by the same MS analysis approach yielded 592 peptides in the 1394/1436 clusters representing 12.1% of the total (Fig. 2). A third survey for the presence of glycopeptides was conducted by performing HILIC enrichment on the total membrane digest sample. As could be expected, the 1394/1436 clusters were greatly enriched, and the presence of potential glycopeptides with Dmass values of ;792 Da were supported (Fig. 2, inset).
To further investigate the clusters, well defined MS1-level spectra of up to 10 peptides exhibiting each Dmass value were inspected (Table S4). In total, the monoisotopic mass was recorded for 64 spectra. In all cases, the Dmass value was calculated to be 1436. 43, 1394.42, 1352.42, or 792.25, and these values were assumed to correspond to the major O-glycans present. The data were then searched again using both Byonic and Mascot with these four Dmass values used as variable modifications specific to Ser or Thr. The searches proved successful in identifying a larger number of glycosylated peptides including almost all of the expected cluster peptides from the Byonic wildcard searches confirming the presence of just four major glycans. In total, 416 different glycopeptide forms were putatively identified with a Byonic score greater than 200 (Table S5). Only 6 of these did not contain a putative Bacteroidetes O-glycosylation motif, and of these, none could be confirmed to be reliable, usually with the incorrect charge state being assigned to the precursor. Therefore, in downstream analyses, only peptides with a putative Bacteroidetes O-glycosylation motif were considered.
Identification of the glycan sequence. The glycan sequence was determined by ion trap resonance-based collision-induced dissociation (CID) spectra of glycopeptides containing the four glycans and confirmed by TFMS cleavage. The proposed sequence of the largest glycan with a Dmass of 1436 is HexNAc-HexNAc(P-Gro-[Ac] 2 )-dHex-Hex-HexA-Hex(dHex). The glycans of Dmass 1436, 1394, and 1352 differed by 42.01 Da, matching an acetyl group (C 2 H 2 O). The CID fragmentation patterns were the same for these glycans, with the 42-Da differences being accounted for in the glycerol moiety ( Fig. 3A to C). Hence, the three glycans are proposed to have either glycerol phosphate, acetyl glycerol phosphate, or diacetyl glycerol phosphate. Accurate mass data were obtained for these glycerol phosphate moieties from oxonium ions in the high resolution HCD scans. In addition to the oxonium ion for HexNAc at m/z 204.086, ions specific to the three glycans were observed at 442.111 (HexNAc-P-Gro-[Ac] 2 ), 400.100 (HexNAc-P-Gro-Ac), and 358.089 (HexNAc-P-Gro). The only glycoform observed with a different monosaccharide sequence was the glycan with Dmass 792, comprising a pentasaccharide of sequence dHex-Hex-HexA-Hex(dHex) (Fig. 3D). Since for the intact glycans, CID was only activated for peptides containing HexNAc, the CID spectra for Dmass 792 peptides were obtained from the acid-cleaved samples.
Since the major glycans were large, the total membrane and soluble fractions were partially deglycosylated with TFMS in an attempt to truncate the glycans. The deglycosylated samples were then fractionated by SDS-PAGE, analyzed by LC-MS/MS and initially searched with Byonic using the wildcard setting. The most frequent Dmass values observed clustered around 499/500 Da, and inspection of the corresponding peptide sequences indicated that they overlapped extensively with the peptides modified with the intact glycans and also contained the same Bacteroidetes O-glycosylation motif ( Table S2). Examination of the MS1-level spectra demonstrated that the true Dmass value was 500.14, corresponding to the Hex-HexA-Hex portion of the intact glycans. The data were searched again with modifications defined in the database for each portion of the intact glycan. TFMS-cleaved glycopeptides were found with Dmass values of 162, 338, 484, 500, 646, and 792 Da matching to various portions of the first five sugars (Table 1). Acid cleavage between the terminal HexNAc residues was not detected. Acid cleaved products with Dmass 1290 Da and 1248 Da were additionally observed corresponding to full-length glycan (1436 or 1394 Da) minus one dHex residue. The most frequent Dmass observed was 500 Da, indicating the strongly preferred cleavage of dHex residues (Table 1).
To enable the characterization of the glycans of acid-cleaved glycopeptides, targeted CID spectra were acquired with the aid of inclusion lists. CID of the Dmass 1290 glycopeptides resulted in a simple, linear fragmentation pathway that matched to the full-length glycan minus the dHex side branch (compare Fig. 4B with Fig. 4A). The 1484 Da fragment (Y 2 in Fig. 4A) was specific to the full-length glycan, suggesting the dHex side branch is located on one of the first two sugars. Its location on the reducing Hex residue was confirmed by analysis of mutants (see below). CID of the smaller acidcleaved glycans of Dmass 646 Da (Fig. 4C), Dmass 500 Da (Fig. 4D), Dmass 338 Da (Fig. 4E), and Dmass 162 Da (Fig. 4F) produced the expected fragment ions, helping to confirm the assigned structure.
Localization of glycosylation sites. All peptides that were confirmed to be glycosylated exhibited an extended Bacteroidetes O-glycosylation motif of (D)(S/T)(A/I/L/V/ M/T/S/G/F/C), the same as found in T. forsythia (33) (Table S2). Since many of these peptides did not have Asn in their sequence or any additional Ser or Thr residues, the glycosylation motifs were the most likely modification sites. Further evidence of modification at these sites was obtained from the "Dmod" scores, which provide the Byonic score of the top matching site minus the second-best matching site. In this study, since the modifications were defined as being linked to Ser or Thr, the score only differentiates between potential Thr and Ser sites. Generally speaking, the ETD or EThcD spectra of glycopeptides gave the best site localization data because in ETD, fragmentation occurs primarily along the peptide backbone, leaving the glycan intact. This mode of fragmentation, however, is generally only optimal for ions of high charge density, ions , 800 m/z and charge state . 13 (34). However, occasionally, the CID spectra produced excellent localization data for doubly-charged glycopeptides, particularly after truncation of the glycans with TFMS. For both intact glycopeptides and acid-truncated glycopeptides, the proportion of glycans assigned to the Bacteroidetes O-glycosylation motif increased with the DMod score ( Table 2). DMod scores above 30 always assigned the site to the motif, providing confidence that glycosylation is indeed occurring at the motif. Higher DMod scores were generally obtained for the acid-cleaved glycopeptides ( Table 2).
While the DMod scores strongly support glycosylation occurring at the Bacteroidetes O-glycosylation motif, they don't show the exact modification site. To show this more directly, four spectra are provided that show the exact location of the glycan within the motifs DSI, DTV, DST, and DTL, respectively ( Fig. 5A to D). In each case, the peptide shown is modified with the most abundant of the acid-cleaved glycans (Dmass = 500 Da). The first three spectra shown are EThcD spectra where fragmentation of the glycan is not evident, and series of c and or z ions demonstrate the modification site ( Fig. 5A-C). The fourth is a CID spectrum (Fig. 5D) where y-ions were observed for both the glycan-cleaved peptide fragments (;y3 -;y11) as well as the peptide fragments that retained the glycan and reveal the modification site (y3, y5-y7).
In addition to detecting glycosylated peptides, the corresponding nonglycosylated peptides were also identified for some sites. Out of the 257 identified sites, only 27 were also found in unmodified form (Table S2). This indicates that on the whole, the level of site occupancy was very high.
Biosynthesis of the glycan. To explore the biosynthesis of the glycan, a panel of mutants was tested by Western blotting of whole cell lysates using antibodies against the Mfa2 protein (PGN_0288) identified to have three glycosylation sites (Table S2). Of the 10 glycosyltransferase mutants plus the waaL ligase and a wzx flippase, only the PGN_1134-1135 double mutant showed an obvious shift in the MW of Mfa2 (Fig. S1), suggesting that one or both of these genes is essential for complete O-glycosylation. To determine the individual contributions of PGN_1134 and PGN_1135, single mutants were created and analyzed by Western blotting. Whole cell lysates of the PGN_1135 mutant produced similar results to the double mutant; however, PGN_1134 produced a smaller shift in the MW of Mfa2 (Fig. 6A). To ensure that the glycosylation defects were specifically due to the targeted genes, the individual mutants were complemented. The complemented strains appeared to produce Mfa2 at the same MW as the wild type (Fig. 6B), confirming that PGN_1134 and PGN_1135 are required for the normal O-glycosylation of Mfa2.  162  338  484  500  646  792  1248  1290  1352  1394  1436  Freq.  36  82  9  265  41  19  10  13  12  22 22 792 Da (D). The first three spectra (A-C) are from the fractionated membrane sample (gel segment #7), and the CID spectra were triggered by the presence of HexNAc (m/z 204.09) in the corresponding HCD spectra. The fourth CID spectrum (D) was taken from the deglycosylated data set (targeted analysis) since it lacks HexNAc. For all spectra, the labeled ions are 2 1 unless shown otherwise. Sugar symbols follow the SNFG as described in Materials and Methods. *, Indicates ions that may be produced by dHex scrambling. To verify that these genes are also required for the O-glycosylation of other proteins and to identify the steps in glycan synthesis blocked by these genes, proteins from whole cell lysates of each mutant strain were separated by SDS-PAGE, in-gel digested, and analyzed by LC-MS/MS. The double mutant and PGN_1135 mutant exhibited similar results, with the largest glycan having a Dmass of 792, which was observed for 124 and 155 nonredundant peptides respectively ( Table 3). The next most common glycan observed exhibited a Dmass of 308, with ;30 peptides suggesting a partial blockage at that step. The largest glycan observed in the PGN_1134 mutant exhibited a Dmass of 995 on 87 peptides; however, a Dmass of 792 was most common, with 157 peptides identified. Inspection of the MS/MS data revealed that peptides with a Dmass of 792 in all mutants fragmented the same as that shown in Fig. 3D suggesting the same pentasaccharide structure. The MS/MS data for peptides with Dmass 308 helpfully confirmed the location of the branched deoxyhexose on the reducing hexose (Fig. 7A). The MS/ MS data for peptides with Dmass 995 were consistent with the same pentasaccharide plus a HexNAc residue (Fig. 7B). Assuming the largest glycans observed in each mutant are the direct substrates of the corresponding enzymes, PGN_1135 is inferred to transfer a HexNAc residue to the pentasaccharide and PGN_1134 is inferred to transfer the terminal HexNAc. Since phosphoglycerol substituents were not identified, it is likely this moiety is transferred at a later step (Fig. 7C).
Glycan heterogeneity. Further Mascot searches of the intact wild-type samples were conducted to determine whether the phosphoglycerol addition was stoichiometric. Small numbers of glycopeptides with Dmass values of 995.33 Da and 1198.41 Da corresponding to the inclusion of one or both HexNAc residues without phosphoglycerol were identified. The abundance (peak intensity) of these glycopeptides was typically ,1% relative to those with phosphoglycerol, suggesting that the modification was nearly stoichiometric. The HPLC retention times of these glycopeptides were always less than their three counterpart glycopeptides substituted with variously acetylated glycerophosphate, confirming genuine sample heterogeneity rather than fragmentation within the mass spectrometer (Fig. S2). Furthermore, acetylation consistently resulted in a slight increase in retention time, again confirming the genuine heterogeneity of acetylation levels (Fig. S2).
The proteins identified. The putative localizations of the 145 glycoproteins identified were inner membrane (IM, 48), lipoproteins (42), periplasm (28), outer membrane (OM, 15), T9SS cargo (4), and uncertain (8) (Table S3). Due to the familiarity of the authors with the OM proteome (20,35), it was immediately apparent that the most abundant OM-associated proteins were absent from the list of identified glycoproteins. To explore this observation further, the theoretical OM proteome and lists of predicted lipoproteins, periplasmic proteins, and IM proteins (36) were examined in detail to determine patterns between abundance, various protein categories, predicted glycosylation sites, and detected glycosylation (Table S6). For the 10 most abundant OM proteins, only three (30%) had predicted O-glycosylation sites (Fig. 8); however, none were identified to be glycosylated, and furthermore the unmodified forms of the peptides bearing the three sites were identified, suggesting that the predicted sites may not be modified at all. In contrast, 80% of the abundant periplasmic proteins, and 100% of the   abundant IM proteins and non-localized lipoproteins, were predicted to be glycosylated, with a total of 13, 38, and 42 predicted sites, respectively (Fig. 8). The overall proportion of proteins glycosylated was substantially lower (55%) for proteins localized to the OM compared to the other locales, which ranged from 71% to 80% (Fig. 8).
Together, the data suggest that the O-glycosylation of OM-associated proteins is nonpreferred, particularly if they are abundant.

DISCUSSION
This is the first detailed report of O-glycosylation in P. gingivalis and the first to directly identify O-linked glycans. This makes O-glycosylation the second protein glycosylation system to be described for this species, after the T9SS-related system, which glycosylates the mature C-terminus of cargo proteins via a peptide linkage (13,14). The glycan was shown to be a heptasaccharide substituted with acetyl glycerol phosphate, which has not been observed in other bacterial glycans to our knowledge. We identified 145 O-glycosylated proteins, which is 40% of the total number (358) predicted for P. gingivalis ATCC 33277 based on the presence of the O-glycosylation motif (27).
The first six monosaccharide classes of the heptasaccharide are almost identical to the first six in the putative B. fragilis O-glycan (37). The only detected differences are in the substitutions of acetyl glycerol phosphate or O-methyl ( Fig. 9A and B). Furthermore, the first four sugars of the B. fragilis O-glycan may be the same as that of Elizabethkingia meningoseptica (Fig. 9C) (38). The most different of the four Bacteroidetes O-glycans is that produced by T. forsythia, which has branches on every monosaccharide, but it still shares a similar trisaccharide unit of HexA-Hex(dHex) (Fig. 9D). The dHex branch in E. meningoseptica is Omethylrhamnose, and the dHex branch in B. fragilis was inferred to be the same (27). While the dHex branch in T. forsythia was reported to be fucose, there is some uncertainty of this because it was originally identified by NMR and thought to branch from the Me-Gal residue (28). Its position branching from the reducing-end Gal was deduced by MS methods (31) and therefore could in theory be a different deoxyhexose. The reducing end sugar is Gal in T. forsythia but Man in E. meningoseptica, so despite commonalities in the first 3-4 sugars, there are also differences. Despite these differences, an antibody that recognizes the "core glycan" of B. fragilis also cross-reacts with glycoproteins from all four species and many other species across all major classes within the Bacteroidetes phylum (27).
Of these four Bacteroidetes O-glycans, the biosynthesis has been best studied in T. forsythia where the roles of five glycosyltransferases have been elucidated (31).  BLAST searches of these enzymes demonstrated the existence of close orthologs for GtfE and GtfL in both P. gingivalis and B. fragilis but not E. meningoseptica (Table 4). In T. forsythia, GtfE was proposed to transfer the branching dHex residue; however, it appears more likely that it transfers the internal Fuc residue (Fig. 9D) since the branching dHex is an optional side-branch that is not present in some native glycans and yet the gtfE mutant produces a pentasaccharide (Gal 1 Dig 1 dHex 1 GlcA 1 Xyl 1 ) as the largest glycan detected (31). If the optional branching dHex residue was transferred by GtfE, full-length glycans (minus dHex) should have been observed in the mutant. This interpretation also fits better with the data as a whole so that the biosynthetic locus encodes for glycosyltransferases transferring adjacent sugars in the outer core (Fig. 9D). We propose that the GtfE orthologs, BF_4306 and PGN_0777, may transfer the putative internal Fuc residue in their respective glycans ( Fig. 9A and B). This putative Fuc is strongly inferred in B. fragilis (27), and proposed in P. gingivalis by homology of structure and biosynthesis (Fig. 9). In T. forsythia, GtfL transfers the Me-Gal residue to the internal Fuc since in the gtfL mutant, the largest glycan observed was the hexasaccharide (31). Similarly, in the PGN_1135 mutant, the largest glycan was the pentasaccharide (Dmass = 792, Table 3), consistent with transferring the first HexNAc residue (Fig. 7C), which may be GalNAc since the homology to GtfL is so strong (Table 4). Similarly, in the PGN_1134 mutant, a hexasaccharide was observed consistent with PGN_1134 transferring the terminal HexNAc (Fig. 7C, Fig. 9A). Interestingly, in B. fragilis mutants lacking the whole "LFG" glycosylation locus spanning BF_4298-4306 or just lacking BF_4306, or when GDP-fucose production was blocked, only a residual O-glycan disaccharide comprising (Me)dHex-Hex was reported (27). However, it is not clear whether larger glycans may also have been present. In our own data (Table 3), the corresponding disaccharide (dHex-Hex) of Dmass 308 was also observed in the tested mutants, suggesting there may be a significant additional blockage at the step of transferring the third sugar. This blockage appears to support the model where the disaccharide core glycan is transferred to the protein first, and then the FIG 8 The O-glycosylation system is biased against abundant OM proteins. The proportion of proteins with predicted O-glycosylation sites is plotted for the 10 most abundant proteins (blue) and all proteins (orange) for each localization category. The first column has a diagonal pattern to indicate that these three proteins may not be glycosylated at all since only the unmodified sites were identified. The line graph (pink) relates to the right-hand axis and shows the number of predicted sites for the 10 most abundant proteins in each category. The complete data are provided in Table S6. outer glycan is subsequently ligated to the core (27). In P. gingivalis, the smallest native glycan observed was the pentasaccharide (Dmass 792), suggesting that the outer core must have a minimum of dHexA-Hex-HexA to be recognized by the ligase and be transferred to the core. The ligase may have a preference for transferring a full-length outer core such that any deletion of the outer core glycosyltransferases leads to some proportion of the glycosylation sites being occupied by the core disaccharide only.
Our data confirm some of the previously identified glycoproteins of P. gingivalis. Omp85, now more commonly known as BamA, is an outer membrane protein that catalyzes the insertion of b-barrel outer membrane proteins into the OM (39). BamA was  (31). Glycosyltransferases predicted or shown to be involved in the biosynthesis are shown. Note that striped sugar symbols are not part of the SNFG but are used here to show the predicted isomeric form of the sugar. previously suggested to be glycosylated by a reduction in MW after TFMS treatment or by mutation of the putative epimerase GalE (23). Here, we confirm that BamA (PGN_0299) is O-glycosylated at 280 Ser, and inspection of the sequence reveals two further predicted sites ( 373 Ser and 31 Thr), helping to explain the significant loss in MW observed after deglycosylation. In addition to BamA, other P. gingivalis proteins involved in the translocation and assembly of Omps (36) were identified to be glycosylated, including associated lipoproteins and chaperones (PGN_0296 and PGN_0301), TamA (PGN_0973 and PGN_0147), and TamB (PGN_0145 and PGN_0148) (Table S3).
Of proteins previously thought to be glycosylated on the basis of carbohydrate staining (25), we established that the peptidyl-prolyl cis-trans isomerase PGN_0743 and the TPR domain protein PGN_1513, both periplasmic proteins, are O-glycosylated (Table S3); however, PGN_0876 and the very abundant Omp41 (PGN_0729) were not found to be O-glycosylated, at least not with the glycans observed in this study.
It is interesting to note than in T. forsythia, the very abundant surface-layer proteins, TfsA and TfsB, are highly O-glycosylated (28). Along with 19 sites in TfsB and 11 sites in TfsA, we recently identified O-glycosylation to be a major feature of T. forsythia T9SS cargo proteins, with 90 additional sites found in 16 cargo proteins (33). In contrast, only 7 sites from 5 P. gingivalis cargo proteins were identified. Importantly, the most abundant cargo proteins, the gingipains, were not identified to be O-glycosylated. Consistent with that, inspection of their sequences revealed a complete absence of the (D)(S/T)(A/I/L/V/M/T/S/C/F/G) O-glycosylation motif. Given the low specificity of the motif, and hence the relative ease of producing the motif through mutation, the lack of O-glycosylation sites in the T9SS cargo proteins suggests that their glycosylation is being selected against. A possible further explanation for this is that P. gingivalis has smooth LPS (contains a long repeating polysaccharide) whereas T. forsythia only has rough LPS (short polysaccharide). The O-glycans of T. forsythia may therefore play an important role as the dominant cell-surface polysaccharide.
O-glycosylation in B. fragilis is speculated to have a stabilizing function since nonglycosylated forms of the glycoproteins were not observed and site-directed mutagenesis of glycosylation sites on a particular glycoprotein appeared to destabilize it (29). It is likely that O-glycosylation in P. gingivalis serves a similar purpose.
The P. gingivalis O-glycosylation system appears to prefer proteins associated with the IM and periplasm, at both protein and glycosylation site levels. Most striking was the bias against glycosylating abundant OM-associated proteins such as the gingipains, the RagAB peptide transporter (40), and the OmpA-like proteins Omp40 (OmpA2) and Omp41 (OmpA1) (41,42) (Table S6). Some of the abundant proteins including RagA, Omp41, PGN_1323, and PGN_0741 had predicted glycosylation sites, but only the unmodified sites were identified consistent with no or little glycosylation (Table S6). Two potential reasons for evolution selecting against the O-glycosylation of these proteins could be (i) the high cost of glycosylating abundant proteins relative to any benefit gained; (ii) adverse alteration of the bulk chemical properties of the OM locale; or (iii) to evade human serum antibody against O-glycans of oral Bacteroidales spp. such as  Tanf_01305 GtfE 53% ID BF4306 50% ID  40% ID, 32% Cov  PGN_1135  Tanf_01300 GtfL 45% ID BF4305 41% ID  27% ID, 49% Cov  PGN_1134 - a Dashes represent no significant hits. Shading represents highly significant matches over the full length of the protein. ID, identity; Cov, sequence coverage; ND, BLAST search not done.
Tannerella forsythia. The evolutionary adaption of O-glycosylation may be relatively fast since the glycosylation motif is not very specific, allowing numerous possibilities for most proteins to evolve an O-glycosylation site through a single site mutation. If true, the affected locale is likely to be the cell surface. The T9SS cargo proteins surround the whole cells to form a surface layer and are glycosylated at the C-terminus with A-LPS (14), which may provide the desired surface chemistry or antigen presentation for P. gingivalis cells. Perhaps the O-glycosylation of abundant cargo proteins and other surfaceexposed proteins has adverse effects on this surface chemistry or antigen presentation, leading to negative selection pressure. This theory would account for the lack of O-glycosylation found in abundant cargo proteins, OM b-barrel proteins, abundant pilins such as FimA and Mfa1, and surface-exposed lipoproteins such as RagB, HmuY, and IhtB (36). Glycosylation of low abundance surface proteins was generally limited to one or two sites and may be tolerated due to their low abundance being of little consequence to the overall surface chemistry.
The main locale of O-glycosylation was the periplasm and in particular the side closest to the IM. For the OM-associated proteins, the highest number of glycosylation sites were found or predicted in proteins with large periplasmic domains such as PorW and PorE (43,44) as well as LptD and TamA (36) (Fig. 10). Similarly, since O-glycosylation does not occur in the cytoplasm, the IM-associated proteins found to be O-glycosylated were also mainly those with large periplasmic domains such as fusion proteins, TamB, LptF, signal peptidases, PorM, penicillin binding proteins, and cell division proteins (Fig. 10, Table S3). In summary, P. gingivalis has two major glycosylation systems, the T9SS-related system targeting cargo proteins localized to the cell surface, and the O-glycosylation system that mainly targets protein domains that are localized to the periplasm.
Construction of the PGN_1010 mutant. The P. gingivalis PGN_1010 (domain of unknown function 5020) mutant was constructed by double recombination of the target gene and the introduction of ermF, as previously described (47). The targeting DNA was constructed as follows. The upstream and downstream regions of the PGN_1010 gene were amplified with two pairs of primers (PGN1010upFw/ PGN1010upRv; PGN1010dwFw/PGN1010dwRv), using the genome of ATCC 33277 as template. The ermF region in the ermF DNA cassette was amplified with ermF-Fw/ermF-Rv using the genome of gtfF (PGN_1668)::ermF mutant (KDP611) as template (16). Using the three purified products, further PCR was performed with a PGN1010upFw/PGN1010dwRv primer pair. The amplified PCR fragment was cloned into pUC118, yielding pUC118-PGN_1010::ermF. Then, pUC118-PGN_1010::ermF was linearized by EcoRI. The linearized DNA was introduced into P. gingivalis ATCC 33277 by electroporation to generate Em r transformant (KDP1106). Transformants were selected on blood agar plates containing 10 mg/mL erythromycin.
Construction of the PGN_1134 and PGN_1135 mutants. To construct the PGN_1134 or PGN_1135 mutants, the downstream region of the PGN_1134 gene or upstream region of the PGN_1135 gene was amplified with PGN1134dwFw/PGN1134dwRv or PGN1135upFw/PGN1135upRv primer pairs, respectively, using P. gingivalis ATCC 33277 genome as the DNA template, and was cloned into pUC118 (TaKaRa, Kusatsu, Japan). The downstream region and upstream region were then purified using lower PstI-SacI sites or lower SphI-BamHI sites from each cloned recombinant plasmid and then exchanged with the corresponding sites of pKD990, which is PGN_1134-1135::ermF in pGEM T-easy vector (16), yielding PGN_1134::ermF or PGN_1135::ermF in pGEM T-easy vector. Finally, each desired recombinant plasmid was digested with SacI and introduced into P. gingivalis ATCC 33277 by electroporation to generate Em r transformant, yielding PGN_1134 (KDP1107) or PGN_1135 (KDP1108) mutants. Transformants were selected on blood agar plates containing 10 mg/mL erythromycin.
Construction of P. gingivalis complemented strains. To create complementation plasmids of the PGN_1134 and PGN_1135 genes from P. gingivalis ATCC 33277, the coding regions were amplified using the corresponding compFw/compRev primer pairs. Each PCR product was cloned into pUC118, yielding pUC118-PGN_1134 and pUC118-PGN_1135.
Cell fractionation. P. gingivalis was grown in broth for 4 days and harvested by centrifugation at 8,000 g. Cells were resuspended in 50-mM sodium acetate, pH 5.3, and lysed using a sonication probe (model CPX 750, Cole Parmer) fitted with a 6.5-mm tapered microtip. The amplitude was set to 40% and the pulser to 1 s on, 2 s off for a total of 30 min. Unlysed cells were removed by centrifugation at 8,000 g. The membrane fraction was separated from the soluble fraction by ultracentrifugation at 100,000 g for 30 min. A portion of the soluble fraction was precipitated with 13% trichloroacetic acid and washed with ice-cold acetone. A portion of the membrane pellet was resuspended in 50 mM sodium acetate, pH 5.3, with the aid of sonication (as above) but at a lower power amplitude (19%) and shorter time to create a suspension of fine particles. 3-(N,N-Dimethylmyristylammonio) propanesulfonate (SB3-14) detergent was then added to 0.25% (wt/vol) and mixed by rotation for 24 h at 4°C. The residual membranes were again pelleted as above, and the supernatant was retained as the extracted membrane sample.
Partial deglycosylation. Portions of the membrane fraction and precipitated soluble fraction were resuspended in 50% acetonitrile-0.1% aqueous trifluoroacetic acid (TFA). The samples were transferred to glass vials, freeze-dried thoroughly, and deglycosylated with trifluoromethanesulfonic acid (TFMS) as previously described (14). TFMS is highly volatile and corrosive and must be used with care in a fume hood. Briefly, samples were placed in an ethanol-dry ice bath for the slow addition of a 50-mL solution comprising 90% TFMS and 10% anhydrous toluene. The samples were then transferred to 220°C and the deglycosylation reaction allowed to proceed for 25 min. The reaction mixture was slowly neutralized on an ethanol-dry ice bath with three volumes (150 mL) of pyridine-methanol-water at a ratio of 3:1:1. Proteins were recovered by the addition of 1 mL distilled water and 200 mL trichloroacetic acid and centrifugation at 14,000 g for 20 min. The precipitates were washed with ice-cold acetone.
SDS-PAGE and in-gel digestion. The extracted membrane sample and the deglycosylated protein samples were separated by SDS-PAGE and fractionated into 8 or 16 gel segments respectively as shown (Fig. 1). Whole cell lysates of the mutant strains were also subjected to SDS-PAGE and divided into 6 gel segments each. The segments were in-gel digested with trypsin as previously described (13) and extracted once with 1% aqueous TFA and once with 50% acetonitrile-0.1% aqueous TFA, both for 15 min in an ultrasonication bath. Extracts were combined, evaporated in a vacuum centrifuge, and dissolved in 2% acetonitrile-0.1% aqueous TFA ready for MS analysis.
Whole sample trypsin digestion and HILIC. Another portion of the membrane pellet was run for a short time (;6 min) into an SDS-PAGE gel using all 10 sample wells, stained with Coomassie blue and digested in-gel with trypsin and extracted, all as previously described (51). The extracted peptides were dried in a vacuum centrifuge and then desalted by solid phase extraction using a C18 cartridge. The peptides were eluted in 80% acetonitrile-1% TFA-19% water ready for HILIC enrichment; however, a portion of the peptides were dried and dissolved in 2% acetonitrile-0.1% aqueous TFA ready for MS analysis. ZIC-HILIC stage tips were constructed as previously described in detail (52). The ZIC-HILIC tip was conditioned with 200 mL of 0.1% TFA followed by 200 mL of 95% acetonitrile. The sample was then loaded onto the tip and washed with 500 mL of 80% acetonitrile-1% TFA-19% water. Finally, the peptides were eluted with 150 mL of 0.1% TFA. Note that the ZIC-HILIC material must remain saturated with solvent at all times throughout the procedure. The eluted sample was dried in a vacuum centrifuge and dissolved in 2% acetonitrile-0.1% aqueous TFA ready for MS analysis.
Mass spectrometry. LC-MS/MS experiments were conducted on a Dionex Ultimate 3000 UHPLC interfaced with an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) as previously described (53), with the following modifications. For the analysis of the whole sample digests (with or without HILIC enrichment), a long gradient was used going from approximately 2%-20% acetonitrile over 90 min and then up to 32% acetonitrile over 45 min. For analysis of the detergent-extracted sample that was fractionated into 8 gel segments, a shorter gradient of 2%-40% acetonitrile over 60 min was employed. For these analyses of intact glycopeptides, a stepped FAIMS method was employed alternating between 225 V and 245 V. Specific glycan fragment ions (204.0867, 366.1396, and 138.0545 m/z) were used to trigger the additional CID, EThcD, and HCD scans (all in the orbitrap) with previously described scanning parameters (53).
For the analysis of acid-cleaved glycopeptides, peptides were eluted over a 30 min gradient from approximately 2%-18% acetonitrile and then up to 32% acetonitrile in a further 10 min. A stepped FAIMS method was employed alternating between 240 V and v60 V, and only HCD spectra were collected using the ion trap. For the collection of ETD and CID spectra, selected samples were analyzed again using the same LC-MS/MS method except that previously identified glycopeptides were preferred by the use of a mass-inclusion list. HCD, ETD, EThcD, and CID scans were all performed in the orbitrap.
To examine the O-glycans present in the mutant strains, the samples were analyzed with a short gradient as for the acid-cleaved samples. For selected gel segments, a method employing only CID fragmentation was used to enable the glycan sequence to be determined. This was followed by the analysis of all the gel segments, employing only HCD fragmentation, and a stepped FAIMS method was employed alternating between 225 V and 245 V.
Peptide identification. Proteins and peptides were identified by searching against the P. gingivalis ATCC 33277 sequence database (GenBank accession: AP009380) (54). All searches were conducted using trypsin with up to two missed cleavages allowed, peptide mass tolerance was set to 10 ppm, fragment mass tolerance to 0.2 Da, and a fixed modification of carbamidomethyl (C). Initially, the raw MS data were searched with Byonic v3.9.6 (Protein Metrics Inc) using the wildcard setting, initially set to 100-2000 Da and later extended to 2500 Da (55). After identification of most of the glycan additions, the data were searched again using specific modifications as follows: common modifications: oxidation (M), 1394.42 (S, T) and 1436.43 (S, T); rare modifications: 792.25 (S, T) and 1352.42 (S, T). A maximum of 3 common and 1 rare modifications were allowed. For the identification of acid-cleaved glycopeptides, Mascot v2.6.2 (Matrix Science, United Kingdom) was used in error-tolerant mode with oxidation (M) set as a variable modification. Only modification fragments specific to the P. gingivalis glycan were added to the list of modifications used for the error-tolerant search. A glycopeptide was considered identified and included in Table S2 if it contained the sequence of the glycosylation motif and was identified by at least one glycopeptide with a Byonic score greater than 200 and -Log 10 (P) . 1 or a Mascot score greater than 30. The false discovery rate (FDR) of identifying individual glycopeptides with these thresholds was ,0.1% for Byonic. Since decoy mode is not compatible with error tolerant mode, the FDR for the Mascot searches was calculated for the most abundant modifications (Hex-HexA and Hex 2 -HexA), which were used as variable modifications. The FDR was insignificant, as no peptides with either modification were identified in the decoy database with a score .30. Where multiple glycopeptides of different sequence or glycan addition were identified for the same site, lower Byonic or Mascot scores were permitted. MS/ MS spectral matches relying on a Byonic score between 200 and 300 were manually checked for the presence of signature low mass ions corresponding to HexNAc, strong matches to b or y ion series, and peaks corresponding to glycan cleavages.
Protein localization. Proteins were localized as follows. Proteins and lipoproteins were localized to the OM or cell surface according to our recently published study (36). Other localizations were according to the "gingimaps" study (56) except when some other knowledge indicated a different localization. Lipoprotein predictions were validated by manual inspection of signals, and those that were not known to be OM (36) were not assigned to either membrane since the lipoprotein sorting signal in P. gingivalis is not yet known.
Data availability. Proteomics data are available via ProteomeXchange with identifier PXD028120.

SUPPLEMENTAL MATERIAL
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