The G. anatis strains used in this work were 12656-12 (β-hemolytic biovar, isolated from chicken liver by Bojesen et al., 2003) and ESV200 (β-hemolytic, isolated in Tepatitlan, Jalisco, Mexico (geographical coordinates: 20° 49′ 0″ north, 102° 44′ 0″ west), in 2009 from hens with respiratory disease by Dr. Edgardo Soriano). Strain purity was checked by streaking on blood agar medium with incubation at 37°C for 24 h. For growth in liquid medium, strains were grown at 37°C for 24 h in brain-heart infusion (BHI) medium (Merck®) with aeration, unless specified otherwise. Precultures were incubated until the optical density at 595 nm (OD₅₉₅) was 1.0 before inoculation in BHI broth in either flasks or microplates (Chantes-Guerra et al., 2022).

Biofilm formation assays were performed as described by Johnson et al. (2013), with some changes as described here, using 24-well cell-culture microplates (TrueLine, LabSource Northlake, IL, United States). For the first inoculum, G. anatis strains were grown in tubes containing 5 ml of liquid BHI medium at 37°C for 18 h with aeration. Bacterial cells were concentrated by centrifugation and adjusted to an OD₆₀₀ of 2.0, and 1:100 dilutions of each strain in liquid BHI medium were prepared. For each dilution, five wells were filled with 1.5 ml of medium, and four of the 24 wells on the plate were reserved for the control, which consisted of BHI inoculated with phosphate-buffered saline (PBS) as described by Cramton et al. (1999). The subsequent assays were performed in triplicate, and the plates were incubated at 37°C under static conditions for 24, 48, 72, and 96 h. After each incubation period, the culture medium and planktonic cells were removed carefully from the wells, and each well was washed twice with triple-distilled water to avoid shearing the biofilm mechanically via water pressure or contact with the pipette or removal of the biofilm from the surface. The plates with biofilms were completely dried by placing them upside down on blotting paper, and each well was filled with 1.5 ml of 0.1% violet crystal, after which the plates were allowed to stand for 30 min at room temperature. The wells were washed four times as described above and dried at room temperature for approximately 45 to 60 min. Two milliliters of acetic acid (33% v/v) were added to each well to solubilize the crystal violet, which was quantified by measuring the OD₆₃₀ in disposable polystyrene cuvettes. To avoid variations in the analysis due to the volatility of acetic acid, the solubilization procedure was performed in five sample blocks in one row at a time to prevent acetic acid evaporation. Normalization of the biofilm data was performed using duplicates of each treatment and each replicate. For each cell culture in the plate used for biofilm quantification, another plate was used for cell quantification by resuspending the biofilms, homogenizing and measuring the OD_600.

For protein analysis, biofilms were recovered from large-volume cultures of G. anatis strains. For this purpose, 150 ml of BHI medium was used to dilute the standardized inoculum at 1:100 as described above. The inoculated medium was placed in 1 L glass bottles placed laying on their sides, and the bottles were incubated statically at 37°C for 24, 48, 72 and 96 h. The medium and planktonic culture were removed carefully by decantation. To recover the biofilms, 2 ml of 50 mM Tris buffer (pH 7.5) was added, and glass beads were used to release the biofilm layer, which was recovered in 15 ml Falcon tubes. The remaining residues were recovered with 500 μl of 50 mM Tris buffer (pH 7.5). The samples were stored at-80°C. The recovered biofilms were subjected to a complex analysis of the changes in their absorbance and protein profiles after treatment with 4 M urea for 30 min, DNase (100 μg/ml), lysozyme (50 μg/ml), proteinase K (50 μg/ml), and pronase E (100 μg/ml). Denaturing 10% polyacrylamide gel electrophoresis (SDS–PAGE) of the biofilm proteins was performed to explore and distinguish planktonic cell growth from adherent cell growth, as reported previously (Lopez-Ochoa et al., 2017).

Total DNA from G. anatis was obtained from broth cultures grown at 37°C for 24 h. A total of 1.5 ml of the culture was centrifuged at 13,000 rpm for 5 min; the cell pellet was recovered, 0.5 ml of lysis buffer (50 mM Tris–HCl (pH 8.0), 10 mM EDTA, 10 mM NaCl) was added, and the sample was homogenized. Then, 20 mg of lysozyme were added and the sample was incubated at 37°C for 20 min; afterward, 40 μl of 1% sodium dodecyl sulfate (SDS) were added, and the sample was homogenized. The lysate was treated with 200 μl of phenol saturated with a solution of Tris and EDTA (or TE) (Sambrook and Russell, 2001), stirred and centrifuged at 13,000 rpm for 10 min. One volume of phenol-TE/chloroform/isoamyl alcohol (25:24:1, v/v) was added to the aqueous phase, and the sample was homogenized and centrifuged at 13,000 rpm for 10 min. The supernatant was recovered, washed twice with chloroform, and centrifuged at 13,000 rpm for 5 min; the upper phase was recovered in a new tube, either 0.1 volume of 3 M sodium acetate (pH 7) or 0.5 volumes of 7.5 M ammonium acetate (pH 7) was added, and the sample was homogenized. Total DNA was precipitated with cold (−20°C) 70% ethanol in water. The mixture was centrifuged at 13,000 rpm for 10 min, the supernatant was discarded, and the DNA pellet was washed with 1 ml of cold 70% ethanol. The DNA was dissolved in 100 μl of sterile distilled water and stored at −20°C until use.

Ribonucleic acid (RNA) was extracted from G. anatis under two different growth conditions, namely, agitation or static conditions, in which the OD₆₀₀ was 1 and 0.5, respectively. To obtain G. anatis ESV200 RNA, a QIAcube Connect (Qiagen® Cat. No./ID: 9002864) was used. In each case, 20 ml of the bacterial culture was centrifuged at 3,500 rpm for 10 min. The supernatant was discarded, and the excess broth was removed with a pipette tip. Then, 500 μl of lysis buffer (10 mM Tris–HCl (pH 8.5), 10 mM EDTA, 10 mM NaCl) was added, and the mixture was homogenized. Then, 500 μl of acid-chloroform-isoamyl phenol was added, and the sample was shaken manually for 2 min and centrifuged at 3,500 rpm for 10 min in a table top centrifuge. The supernatant was transferred to another tube, and 200 μl of 7.5 M ammonium acetate and 500 μl of acid-chloroform-isoamyl phenol were added. The sample was then mixed vigorously for 2 min before centrifugation at 3,500 rpm for 10 min. The aqueous phase was transferred to another tube and washed twice with chloroform. After centrifugation, the aqueous phase was precipitated with 2 volumes of cold absolute ethanol and allowed to precipitate overnight. To recover, the RNA mixture was centrifuged at 3,500 rpm for 10 min, and the pellet was washed twice with cold 70% ethanol. Once dry, the pellet was dissolved in 200 μl of diethyl pyrocarbonate (DEPC)-treated water. The nucleic acid purity was estimated by spectrophotometry based on its absorbance at 260, 230, and 280 nm; RNA integrity was checked by analyzing the electrophoretic profile in a 1% agarose gel, and the samples were stored at −80°C until use.

Bacteria were cultivated on 10% sheep blood agar at 37°C and incubated overnight in a candle jar. The bacterial inoculum was prepared by cultivation of each strain for 14 h in BHI broth.

Specific pathogen-free chickens (Alpes I, Puebla, México) of mixed sexes were divided into two groups of 5 chickens each. The chickens were individually labeled and injected intravenously with 0.2 ml of G. anatis inoculum (approximately 1 × 10⁸ viable cells). The negative control group was inoculated by intravenously injecting 0.2 ml of BHI medium.

Clinical examination, morbidity and mortality data were recorded for each bird group at 7 days post-infection (dpi). Subsequently, the animals were euthanized, and the skin and mucosa (crest, chin, nostrils, eyes, skin, eyelids), respiratory system (infraorbital sinuses, trachea, syrinx, lungs), digestive system (oral cavity, esophagus, craw, proventriculus, gizzard, liver, pancreas, duodenum, jejunum and ileum, blind, rectum, tonsils C, palatal fissure), circulatory and hemolymphatic apparatus (heart, spleen, pouch of Fabrizio, thymus), genitourinary apparatus (testis, ovary, oviduct, follicle, kidneys, ureters), and skeletal muscle system (bones, joints, plantar tissue, bone marrow) from all birds were examined for histopathological signs, followed by bacterial isolation. Samples of tissues with lesions were collected with sterilized cotton swabs, streaked on blood agar plates, and incubated for 24 h at 37.5°C in a microaerophilic environment.

For histopathological analysis, the samples were placed in 10% neutral buffered formalin and processed, sectioned, and stained as described elsewhere. Microscopic lesions were scored on a scale of 0–3: 0 = normal, 1 = minimal changes, 2 = mild changes, and 3 = severe changes. When the observation fields of a single section had lesions with different severities, the lesion degree predominant in 50% or more of the observation fields was used for the aforementioned scoring. The average score for each group was determined by adding individual scores and dividing the total score by the number of slides examined.

The Kruskal–Wallis test was used to assess the differences in the median scores of microscopic lesions between the groups, and differences were considered significant at p = 0.05, as reported elsewhere. Chi-square tests were used to examine the differences between the groups in isolation (presence of the inoculated strains in either sinus) and morbidity rates (percentage of birds that showed any clinical signs during the 7-day observation period), with differences being considered significant at p = 0.05 (Zepeda-Lopez et al., 2010).

To analyze the changes in the transcriptional expression of the fimbrial and adhesin genes of both the 12656-12 and ESV200 strains by end-point RT–PCR, we used the UMN179 genome sequences (http://www.ncbi.nlm.nih.gov/genome/3653?project_id=66567 from Johnson et al., 2011) of the gene Flf1_a to design the primer pair Fifa7upp and Fifa7rev that amplified a 236-bp fragment (Table 1). The genome sequences of the 12656-12 strain¹ were used for subsequent primer design: for the Flf3_b, Flf_b, Flf3, Flf1, and, Flf-related genes, pairs of primers were designed, whose names, primer sequences and sizes of the PCR amplicons are shown in Table 1. It was not necessary to make more primer designs because the size of the synthesized amplicons was appropriate to carry out the end-point PCR and qRT–PCR (see below). As a positive control, total DNA from G. anatis 12656-12 was used for PCR, and as a negative control, a reaction without reverse transcriptase was performed.

The reaction mixture for endpoint PCR contained 10 ng of total DNA from Gallibacterium strains as template, 0.3 μM each DNA oligonucleotide primer, 10 mM Tris–HCl (pH 8.4), 1.5 mM MgCl₂, 0.24 mM each dNTP and 0.3 U of Platinum TaqDNA polymerase (Invitrogen, Thermo Fisher Scientific. Waltham, MA, United States). The mixture was homogenized and treated as follows: denaturation for 5 min at 95°C, followed by 30 cycles of the appropriate annealing temperature and 72°C for elongation in a T100 thermal cycler (Bio-Rad, Hercules, CA, USA). The amplified products were electrophoresed in a 0.8% agarose gel, stained with ethidium bromide solution (2 μg/ml) and observed under ultraviolet light.

To analyze the changes in transcriptional expression by RT–PCR, the Ct cycle method was used. Using the constitutively expressed genes as a reference, we chose the hypothetical 901-bp rpoA gene, which encodes the RNA-polymerase alpha-subunit RpoA, as well as the 2,106-bp putative fusA gene from G. anatis UMN179, which encodes a hypothetical elongation factor G (EFG). The primers for these two genes and the primers used for analysis of the expression of the fimbrial genes are listed in Table 1. Due to the small size of the amplicons and the fact that the primers were well evaluated for their amplification efficiency, the same primers were used in the end-point PCR and quantitative RT-PCR. We expected that the transcripts of the housekeeping genes would show stable levels of expression under different conditions for normalization, and we chose to apply the formula described by Pfaffl (2001).

Real-time PCR was performed in an IQTM5 Multicolor Real Time PCR Detection System (Bio-Rad). For each qPCR sample, three technical replicates and two biological replicates were examined. For each primer pair, a no-template control (NTC) was included to detect the formation of primer dimers. The reaction mixture was prepared at room temperature as follows: 1 μl of cDNA, 1 μl of 10 μM forward primer, 1 μl of 10 μM reverse primer, 10 μl of SYBR Green PCR master mix (Thermo Scientific) and 7 μl of water. The SYBR Green mix already contains DNA polymerase, SYBR Green and a passive reference dye (ROX), along with the required reaction components. Individual concave-cap tubes were used. The reaction was carried out according to the supplier’s instructions: 1 cycle of 95°C/10 min; 40 cycles of 95°C/15 s, 60°C/15 s; 72°C/30 s. For the denaturation curve, 60 cycles at 65°C were performed, with the time increasing by 5 s in each cycle. Once the experimental Ct values were obtained, the dynamic range was calculated using log₁₀ dilutions for the test, covering a range of 10⁰ to 10⁸ molecules. The efficiency of the qRT-PCRs was determined, and those with efficiencies greater than 96% and an R² value greater than 0.86 were considered valid. The Supplementary material includes graphs showing the expression of some genes.

The total DNA of G. anatis ESV200 was obtained to carry out nucleotide sequencing of the genome. Two sequencing technologies, namely, the NextSeq500 platform from Illumina® and the Sequel System from PacBio®, were used to obtain a unique chromosomal molecule. The software packages used to join reads into a single chromosomal sequence were FASTQC 11.9 (Wingett and Andrews, 2018), Velvet 1.0.19 (Zerbino and Birney, 2008), Spades 3.15.5 (Bankevich et al., 2012), and Canu 2.3 (Koren et al., 2017). The gene content in the genome of the ESV200 strain was explored by comparing the information deposited in GenBank under accession number CP114281 (WGS- Project-CP114281.1) and three other records for strains UMN179 (CP002667), F149 (JPHN01000000) and 12656-12 (AVOX00000000). The genomes included correspond to the most well-studied strains of G. anatis, and because the pathogenicity was compared with reference to the virulent strain 12656-12, UMN179 is the only completely circularized genome. Complete chromosomes were compared (Dhillon et al., 2013) with Rapid Annotation using Subsystem Technology (RAST; Aziz et al., 2008; Overbeek et al., 2014) and displayed in a graphical representation prepared with the Easyfig program (Sullivan et al., 2011). Additionally, a review of the gene annotation obtained by the GenBank pipeline was performed. The contents of various RNA types were verified using Basic Local Alignment Search Tool - BLASTn (www.ncbi.nlm.nih.gov; Madden et al., 1996) and tRNAscan-SE 2.0 (Chan et al., 2021). The phylogenetic analysis of the four fimbrial operon genes was performed as described Kudirkiene et al. (2014), although the sequences of the genes encoding the Usher chaperone, the major protein, and the adhesin were separated for a more appropriate comparison. Clusters were constructed by comparison of G. anatis gene sequences from operons with Clustal Omega (Madeira et al., 2022): the sequences included here were from the G. anatis ESV200, UMN179, and 12656-12 strains. The phylogenetic analysis of G. anatis ESV200 was performed by comparing eight concatenated housekeeping genes (adk, atpD, fumD, gyrB, inf2, mdh, recN, and tdh) previously used as multilocus sequence typing (MLST) markers in G. anatis (Johnson et al., 2013). The neighbor-joining method was used to draw the dendrogram using the online programs Clustal Omega,² Kalign (Lassmann, 2019) and iTol (Letunic and Bork, 2021). A phylogram was constructed with or without a root of two MLST sequences from Avibacterium endocarditis 20186H4H1 and A. gallinarum NCTC11188 strains (Accession numbers: NZ_PQVI01000000 and NZ_PQVJ00000000.1, respectively). MEGA v.11 software was used to assess the confidence of the phylogram of G. anatis MLST sequences through a bootstrapping test with 500 resamples.

Usually, G. anatis 12656-12 and ESV200 strains cultured in the same conditions show different turbidity or clumping phenotypes in liquid BHI medium, and 12656-12 strain cultures agglutinate after a few minutes if kept under static conditions; on the other hand, ESV200 strain cultures form stable dispersed suspensions, and this behavior could be a result of inoculum size, aeration and biofilm capacity. To analyze the development of turbidity and cell agglomeration, two types of cultures were prepared by incubation at 37°C, one agitated at 200 rpm to force the formation of a homogeneous suspension and the other unagitated. The shaken cultures consistently exhibited an OD₆₀₀ of 1.9 at 24 h, while the static cultures showed a maximal OD₆₀₀ of 1.9 at 96 h for G. anatis 12656-12 and a maximal OD₆₀₀ of 1.2 at 24 h for G. anatis ESV200. Two conditions were established for comparative quantitation of gene expression in G. anatis at different culture times, as shown below. The results for the quantitation of biofilm formation are depicted in Figure 1, showing an increase in biofilm quantity for both strains at 48 vs. 24, 72, and 96 h. As depicted in Figure 1A, strain ESV200 showed maximal production of biofilm at 48 h, which is 25% more than the amount of biofilm produced by the 12656-12 strain. However, the ESV200 biofilm was unstable, showing a drastic decrease of approximately 80% between 48 and 72 h. The homogeneous suspension and the minor quantity of biofilm formed confirmed the predominant growth of ESV200 in liquid culture medium as a major planktonic suspension, with a temporary exception at 48 h. In contrast, the 12656-12 strain was clumpy in liquid BHI medium and maintained the biofilm structure for a long time until the cell density was double that of ESV200 at 72 h. These differences observed for the ESV200 strain cultures indicated that biofilm formation was turned on in a transient manner after disintegration of the biofilm structure but did not indicate an inability to produce a biofilm. The 12656-12 strain showed a robust biofilm.

The protein profiles of the planktonic and biofilm cultures were studied using SDS–PAGE to determine the importance of this type of cellular component in the formation of aggregates or in maintaining cellular dispersion. The protein patterns of bacterial growth of both strains under agitation showed higher levels of recoverable and visible proteins in the SDS–PAGE with differences in the levels of some proteins, which suggests that the proteins could be relevant in the adherence or dispersibility of both strains, phenotypes that differed between these two strains (Figure 1B). Therefore, it can be inferred that the ESV200 strain, because of its proteins, was more compatible with the aqueous medium because it remained dispersed for a longer time. Cultures of the same strain with planktonic cells and biofilms also showed differences in the expression patterns of different proteins. The ESV200 strain showed five protein bands with increasing concentrations in the biofilm structure at 37°C. The concentrations of the visible proteins decreased at 42°C, indicating regulation mediated by temperature.

In G. anatis 12656-12, an increase in the levels of more than 6 accumulated protein bands in the adherent state was observed (the arrows show the 5 most evident bands of less than 100 kDa). This profile was maintained at 37 or 42°C of incubation; therefore, this variation in the culture conditions led to little alteration in 12656-12 strain metabolism.

As it has been reported that bacterial biofilms represent a state of cell agglomeration with a complex matrix, the biofilm was treated for 48 h with 4 different enzymes (two proteases, DNase and lysozyme) and urea (Figures 1C,D) to explore the changes in protein profile in the biofilm matrix. These treatments would increase the turbidity of the biofilm suspension if they separated the cell clumps. Figure 1C shows the change in cell agglomeration caused by DNase, proteinase K and pronase E. In samples from the ESV200 biofilm, DNase and proteinase K treatments increased the turbidity measured at 630 nm, while pronase E reduced the turbidity. For the 12656-12 biofilm, only treatment with DNase disintegrated the agglomerates and induced an increase in the OD; the proteases used did not have this effect. The protein profile was not altered by DNase, lysozyme or urea, although the separation of the proteins was slightly improved with respect to the profile of the proteins obtained by simple resuspension in the 2X loading buffer used for SDS–PAGE.

Because the two strains of G. anatis investigated had different agglomeration behaviors, their pathogenic capacity was analyzed in groups of five birds at 8 weeks of age, and relatively similar results were obtained when the birds were inoculated intravenously with 0.2 ml of fresh culture. The symptoms appeared on the 4th day postinoculation, although there was greater decay in the group inoculated with ESV200 (Table 2). There was no evident damage to the respiratory system of birds inoculated with any of the strains during necropsy. However, a higher rate of damage was observed in the organs (liver, spleen or kidney) of the birds inoculated. Damage to the heart was observed in birds inoculated with the G. anatis 12656-12 strain but not in birds inoculated with the ESV200 strain. The injected bacteria were unable to be recovered from the most frequently damaged organs.

As changes in bacterial aggregation have been observed through treatment with proteases and fibrillar structures by electron microscopy of G. anatis biofilms (Vaca et al., 2011; Salgado Lucio et al., 2012), we asked how some extracellular proteins of G. anatis are expressed, as they could form cellular junctions within the biofilms or bridges of cells with solid surfaces, so the abundances of the mRNA of the fimbrial genes flf1_a, flf3_b, flf_b, flf1_D, flf3_D, and flf_D (Figure 2A) were determined at the transcriptional level (Figure 2B). Absolute quantitation of the transcripts by qRT–PCR was performed by using standard curves prepared for each of the genes; the expression of each transcript was different, and the flf3 gene primers did not amplify any fragment from ESV200 DNA, either due to variations in the target sequence of the primers or due to the absence of the gene in the genome of the ESV200 strain. Another gene that is poorly expressed in G. anatis ESV200 is flf_D. However, the structural and adhesin genes of the flf_b operon of ESV200 correspond to two different operons in G. anatis 12656-12; the homologous but more divergent adhesin (89% identity) is part of the flf3_b operon, and the remaining three genes, including the gene encoding the major fimbrial protein (98% identical), are present in flf_b. According to the expression analysis of the fimbrial genes in strain 12656-12, higher expression was induced under static conditions (Figure 3).

The relative expression of fimbrial mRNAs with respect to the expression of mRNA of the RNA polymerase gene (Figure 3A) showed a constant expression level in the 12656-12 strain under static conditions, and the expression levels of the three mRNAs of the three fimbrial genes (0.5 at 4X) and the three mRNAs encoding adhesins (1 to 7X) changed under agitation conditions.

For the ESV200 strain, 4X greater expression of the mRNA encoding the Flf1_a protein was observed, regardless of the culture conditions, without the expression of Flf1_D. For the major protein Flf3_b, there was no expression in the ESV200 strain, and for adhesin, low expression was observed, perhaps as an important housekeeping gene. For the major protein Flf_b in ESV200, vestigial expression was observed, while increased expression (4X) of Flf was observed. In a comparative analysis of expression of the target genes with that of the housekeeping gene encoding EFG (Figure 3B), basal expression was also observed under aeration conditions in the 12656-12 strain, while the expression of the fimbrial proteins increased from 2X to 5X under static conditions; the expression of adhesins increased from 3X to 9X. In strain ESV200, there were no important changes in the expression of the majority fimbrial protein or adhesin, although under aeration, low expression of the adhesin Flf_D was observed (Figure 3).

To further understand the form of expression of the fimbriae in G. anatis ESV200, the sequence of the ESV200 genome was obtained, and information on the fimbrial operons was extracted. This information was compared with that of the genomes of strains UMN179, 12656-12 and F149^T. The ESV200 strain genome was found to contain two fimbrial operons, while the other three strains possessed three operons (Figure 2). These operons are based on a secretion system that involves the Usher chaperone located on the outer membrane of the bacterium. This chaperone is the largest protein of the chaperone/Usher system that, together with another smaller periplasmic chaperone encoded by the fimbrial operon, is involved in the secretion of the fimbrial structure. The sequence of the usher chaperone gene of the ESV200 strain was compared with others (Figure 4), and it was found that, in addition to the open reading frame (ORF) for the usher gene, there was an internal reading frame in one of the usher genes in strains 12656-12, F149^T and UMN179 (Figure 3C), but this reading frame was absent in the ESV200 strain. The absence of this extra ORF may be related to the lower biofilm production by this strain.

Nine integrase-related amino acid sequences, ranging in size from 60 to 415 amino acids, were deduced in silico from the G. anatis ESV200 genome. A 168 amino acid sequence was annotated as an integrating regulator; therefore, the regulator size selection criterion was used to separate the integrase sequences into two categories. Thus, five large sequences remained as integrases (252 to 415 aa) and the four short ones as possible integration regulators (60 to 170 aa). The short sequences showed a conserved amino-terminal domain of 60 amino acids, although the integrase regulator had more significant differences from the other short proteins. Essential amino acids that are part of the active site for integration, including tyrosine, were identified in proteins from 252 to 415 aa. Three amino acid sequences related to integrases delimited a 539.6 kb fragment. This sequence is flanked at the ends by two segments of mobile genetic elements, at one end an episome type and at the other a prophage element, with 50.5 and 46.3 kb, respectively. The remarkable aspect of the chromosome fragment is that it contains chromosome, phage, and plasmid replication gene clusters. Mobilization genes and plasmid transfer. Genes that can provide resistance to antimicrobial substances as ejection pumps. Genes for RNA polymerase peptide biosynthesis. Genes involved in ribosome biosynthesis, such as ribosomal RNA and ribosomal proteins. Genes for the biosynthesis of aminoacyl-tRNA synthetases. Genes for the biosynthesis of chaperones for the export of extracellular proteins. Genes for fimbriae biosynthesis and control of biofilm formation. By size, this segment resembles a bacterial minichromosome. However, due to the lack of essential genes, it has an integron-type structure, similar to those called Maverick. This chromosome segment is equivalent to a domain called “D1,” composed of 22% of all genes. The “D2 and D3” domains, delimited by the mobile element regions, contain 25.2 and 52.8% of all genes, respectively. The D1 domain contains 5% hypothetical and DNA mobility genes. Regarding the exclusive DNA mobility genes, D1 contained 3.1%, D2 6.1%, and D3 0.94%. This means more significant potential for DNA mobility in the small domains than in the large domain of the G. anatis ESV200 chromosome.