Evolutionary genetics of the capsular locus of serogroup 6 pneumococci

The evolution of the capsular biosynthetic (cps) locus of serogroup 6 Streptococcus pneumoniae was investigated by analyzing sequence variation within three serotype-specific cps genes from 102 serotype 6A and 6B isolates. Sequence variation within these cps genes was related to the genetic relatedness of the isolates, determined by multilocus sequence typing, and to the inferred patterns of recent evolutionary descent, explored using the eBURST algorithm. The serotype-specific cps genes had a low percent G+C, and there was a low level of sequence diversity in this region among serotype 6A and 6B isolates. There was also little sequence divergence between these serotypes, suggesting a single introduction of an ancestral cps sequence, followed by slight divergence to create serotypes 6A and 6B. A minority of serotype 6B isolates had cps sequences (class 2 sequences) that were approximately 5% divergent from those of other serotype 6B isolates (class 1 sequences) and which may have arisen by a second, more recent introduction from a related but distinct source. Expression of a serotype 6A or 6B capsule correlated perfectly with a single nonsynonymous polymorphism within wciP, the rhamnosyl transferase gene. In addition to ample evidence of the horizontal transfer of the serotype 6A and 6B cps locus into unrelated lineages, there was evidence for relatively frequent changes from serotype 6A to 6B, and vice versa, among very closely related isolates and examples of recent recombinational events between class 1 and 2 cps serogroup 6 sequences.     

Immunochemical studies and genetic background of two Neisseria meningitidis isolates expressing unusual capsule polysaccharide antigens with specificities of both serogroup Y and W135

We described 2 unusual Neisseria meningitidis strains isolated from epidemiologically unrelated invasive meningococcal disease cases in Ontario, Canada. Both isolates have features typical of serogroup Y N. meningitidis: are of serotype 2c, are of the multi-locus sequence types typical of the serogroup Y strains in Canada, and are genotyped as serogroup Y based on a previously described PCR-ELISA method that detects the serogroup-Y-specific siaD gene. However, both strains were poly-agglutinable in both anti-Y and anti-W135 antisera. Further studies on 1 of these 2 isolates showed the presence of glucose and galactose as well as sialic acids in its purified capsular polysaccharide, suggesting the presence of both serogroup Y and serogroup W135 polysaccharides. Rabbit antisera produced to this strain contained antibodies to both purified serogroup Y and serogroup W135 capsular polysaccharides. Absorption experiments with either serogroup Y or serogroup W135 bacteria confirmed the presence of antibodies to these 2 different polysaccharides. DNA sequencing of the cps operon from both isolates revealed a siaD gene with 99.7% homology to the published siaD sequence from a serogroup Y strain but with 3 point mutations that all resulted in amino acid changes. How these strains may affect results of routine surveillance, PCR diagnosis, and immuno-protection by vaccination are discussed.     

Characterization of Neisseria meningitidis strains isolated from invasive meningococcal disease cases in Canada in 2001

With the recent introduction of polysaccharide-protein conjugated vaccines for the control of serogroup C meningococcal disease and the emergence of different variants of serogroup C meningococci, it is likely the epidemiology of meningococcal disease in many countries may be affected. We have therefore analysed and reported the characteristics of Neisseria meningitidis strains collected in 2001 from the Canadian surveillance program on invasive meningococcal disease. Only strains collected from normally sterile clinical sites of patients were studied. Of the 289 isolates obtained from individual patients, 173 (59.9%) were serogroup C, 76 (26.3%) were serogroup B, 30 (10.4%) were serogroup Y, and 10 (3.5%) were serogroup W135. Ninety-six percent of the serogroup C isolates belonged to the ET-15 clone, with an additional 2.3% belonging to other electrophoretic types within the ET-37 clonal complex. Different antigenic variants of the endemic serogroup C ET-15 clone were responsible for localized outbreaks in different parts of the country. One novel variant with the antigenic composition of C:2a:P1.1,7 was reported in two provinces, Quebec and Ontario. Eighteen percent of the meningococci isolated from patients in Ontario belonged to serogroup Y, compared with only 8% in the rest of Canada. The current data highlight the importance of strain characterization by serogroup, serotype, and serosubtype antigens in providing useful information for the surveillance of meningococcal disease in Canada.     

Conserved virulence of C to B capsule switched Neisseria meningitidis clinical isolates belonging to ET-37/ST-11 clonal complex

Capsule switching in Neisseria meningitidis is thought to occur by horizontal DNA exchange between meningococcal strains. Antigenic variants may be generated by allelic replacement of the siaD gene; the variants may then be selected by specific immunity against the capsular antigen. There were several vaccination campaigns against serogroup C in France in 2002, following an increase in the prevalence of invasive isolates of serogroup C of the phenotype C:2a:P1.5 and C:2a:P1.5,2 belonging to the ET-37/ST-11 clonal complex. We evaluated the emergence of capsule variants by the detection of B:2a:P1.5 and B:2a:P1.5,2 meningococcal isolates of the ET-37/ST-11 clonal complex. These isolates were significantly more frequent after the year 2002. Pulsed field gel electrophoresis profiles of the serogroup B (ET-37/ST-11) isolates differed from that of serogroup C (ET-37/ST-11) isolates by the bands that harbor the siaD genes responsible for the serogroup specificity. However, serogroup B and C, ET37/ST-11 isolates both express similar virulence as assessed from colonization and invasiveness in a mouse model. Our results indicate that capsule switching events within the same clonal complex can arise frequently with no alteration in virulence. This justifies an enhanced system of surveillance by molecular typing of such isolates, particularly after serogroup-specific vaccination.     

Comparative Genetics of Capsular Polysaccharide Biosynthesis in Streptococcus pneumoniae Types Belonging to Serogroup 19

The genetic basis for the structural diversity of capsule polysaccharide (CPS) in Streptococcus pneumoniae serogroup 19 (consisting of types 19F, 19A, 19B, and 19C) has been determined for the first time. In this study, the genetic basis for the 19A and 19C serotypes is described, and the structures of all four serogroup 19 cps loci and their flanking sequences are compared. Transformation studies show that the structural difference between the 19A and 19F CPSs is likely to be a consequence of differences between their respective polysaccharide polymerase genes (cps19aI and cps19fI). The CPS of type 19C differs from that of type 19B by the addition of glucose. We have identified a single gene difference between the two cps loci (cps19cS), which is likely to encode a glucosyl transferase. The arrangement of the genes within the cps19 loci is highly conserved, with 13 genes (cps19A to -H and cps19K to -O) common to all four serogroup 19 members. These cps genes encode functions required for the synthesis of the shared trisaccharide component of the group 19 CPS repeat unit structures. Furthermore, the genetic differences between the group 19 cps loci identified are consistent with the CPS structures of the individual serotypes. Functions have been assigned to nearly all of the cps19 gene products, based on either gene complementation or similarity to other proteins with known functions, and putative biosynthetic pathways for production of all four group 19 CPSs have been proposed.     

Occurrence of selected loci of the cps region for capsule K1 or K2 synthesis in epidemic strains of Klebsiella pneumoniae isolated from infants in Poland

Number of 60 epidemic strains of K. pneumoniae and 15 isolated occasionally from stool samples were tested for presence of 4 and 11 selected loci of the cps cluster of K1 and K2. Following open reading frames (ORFs): 1, 2, 3, 4, 7, 9, 10, 14, 15 (gnd) of cps K2 strain Chedid and genes magA, gmd, wzc, wca of cps K1 strain DTS were searched by PCR in tested and reference strains O1:K1 A5054 and O1:K2 B5055. The ORFs 1 to 3 and ORF15 were detected in both the reference and epidemic strains as well as in the greatest majority of the occasional isolates. Thus, such ORFs were found K. pneumoniae cps common domains, while tested ORFs 4 to 14 were observed in strain B5055 and 11 epidemic isolates from patients of the same hospital ward. Only exception was a single strain O3 occasionally isolated from faeces. The tested genes of cps K1 were detected only in strain A5054 and in two O3 occasional isolates from faeces. Interestingly, these genes as well ORFs 4 to 14 were detected together in the appropriate reference and tested strains with only two exceptions. Therefore, the cps sector occupied by ORFs 4 to 14 was found as group-specific domain. The occurrence ratio of cps K2 group-specific loci among epidemic strains from infants was 18%, while the K1 group-specific loci were absent.     

Infectious molecular clone of a recently transmitted pediatric human immunodeficiency virus clade C isolate from Africa: evidence of intraclade recombination

Although human immunodeficiency virus type 1 (HIV-1) clade C continues to dominate the pandemic, only two infectious clade C proviral DNA clones have been described (N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T. N. Dhole, Y. Takebe, M. Matsuda, and M. Tatsumi, AIDS Res. Hum. Retrovir. 15:1321-1324, 1999; T. Ndung'u, B. Renjifo, and M. Essex, J. Virol. 75:4964-4972, 2001). We have generated an infectious molecular clone of a pediatric clade C strain, HIV1084i, which was isolated from a Zambian infant infected either intrapartum or through breastfeeding. HIV1084i is an R5, non-syncytium-inducing isolate that bears all known clade C signatures; gag, pol, and env consistently mapped within clade C. Interestingly, gag resembled Asian isolates, whereas pol and env resembled African isolates, indicating that HIV1084i probably arose from an intraclade recombination. As a recently transmitted clade C strain, HIV1084i will be a useful vaccine development tool.     

Recent emergence of serogroup C meningococcal disease in Greece

The number of cases of meningococcal disease reported to the Meningitis Reference Laboratory in Athens rose dramatically in 1996-1997. The aims were (1) to determine if the increase was due to introduction of new strains, (2) to assess the geographic and age distribution of the cases, (3) to compare antibiotic sensitivity patterns of the current isolates with strains from the early 1990s. In 1993-1994, 15/19 (74%) of the cases for which information on age was available were in children < or = 5 years; in 1995-1997, 80/179 (45%) of cases were in children < or = 5 years and 99 (55%) in the older age range (P < 0.02). From 593 cases in 1993 1997, 214 (36%) isolates were available for characterisation. Serogroup B was predominant in the early 1990s, but by 1997, serogroup C accounted for 46/72 (64%) of isolates and serogroup B for 25/72 (35%). Serogroup B was predominant in children < or = 5 years (44/78, 56%) but only 19/99 (18%) of older children and adults (P=0.0000005). Sulfonamide resistance decreased from 10/22 (45%) in 1993-1994 to 27/192 (14%) in 1995-1997 (P<0.01). Multilocus enzyme electrophoresis of 70 strains obtained during this period identified the epidemic ET-15 clone in 24 (34.3)%. The profiles of the Greek ET-15 isolates were identical to C:2a:P1.2(P1.5) strains responsible for the epidemic in the Czech Republic which began in 1993. This genotype was not found in Greek strains isolated prior to 1993. We conclude that the increase in meningococcal disease is due to introduction of the epidemic serogroup C:2a:P1.2(P1.5) strain responsible for disease in the Czech Republic and Canada.     

HmbR, a hemoglobin-binding outer membrane protein of Neisseria meningitidis, undergoes phase variation

Neisseria meningitidis uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the hmbR expression pattern in strains in which the hpuB gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some hpuB mutants is suggestive of phase variation of hmbR. The length of the poly(G) tract starting at position +1164 of hmbR absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring the hmbR reading frame. Inactivation of hmbR in these strains resulted in a loss of Hb utilization, demonstrating that the change in the hmbR gene was responsible for the phenotypic switch. The switching rates from hmbR phase off to phase on were approximately 5 x 10(-4) in four serogroup C strains, 2 x 10(-2) in the serogroup A isolate, and 7 x 10(-6) in the serogroup B isolate.     

Characterization of serogroup C meningococci isolated from 14 provinces of China during 1966-2005 using comparative genomic hybridization

Neisseria meningitidis is a major cause of bacterial meningitis and septicemia worldwide. In China, serogroup A strains were responsible for over 95% of the cases, while serogroup B strains were mainly the cause of localized outbreaks and sporadic cases. Before 2003, serogroup C strains were only recovered from a few sporadic cases. However, a sudden increase in the number of cases due to serogroup C strains occurred during 2003-2005 in Anhui Province, China. Many cases were found in other provinces at the same time. Multilocus sequence typing (MLST) results indicated that the unique sequence type 4821 clone meningococci, a new hyper-virulent lineage, was responsible for the serogroup C meningitis outbreaks. We have completed the project of sequencing the whole genome of the Chinese N. meningitidis serogroup C representative isolate 053442. We fabricated a whole-genome microarray of N. meningitidis isolate 053442 and analyzed the genome composition differences among 81 serogroup C isolates which were isolated from 14 provinces of China during 1966-2005. The comparative genomic hybridization (CGH) result shows that the genome compositions of nearly all serogroup C isolates are similar to that of 053442. The products of many absent open reading frames (ORFs) are conserved hypothetical proteins. The results will provide a valuable resource from which one can analyze the genome composition and genetic background of serogroup C meningococci in China.     

Generation of capsule-deficient Neisseria meningitidis strains by homologous recombination

Capsule-deficient mutants of Neisseria meningitidis serogroup B strain B1940 were constructed by allelic replacement using the plasmids pMF120 and pMF121, which contain the flanking regions of the gene locus for the biosynthesis pathway of the group B meningococcal capsular polysaccharide. Southern blot analysis of chromosomal DNA of the capsule-deficient meningococcal strains confirmed the generation of large deletions in the chromosomal cps gene complex. The same strategy proved useful in constructing meningococcal strains with capsular types A, C, W135, Y and Z.     

Comparative structural and molecular characterization of Streptococcus pneumoniae capsular polysaccharide serogroup 10

Streptococcus pneumoniae serogroup 10 includes four cross-reactive capsular polysaccharide (CPS) serotypes (10F, 10A, 10B, and 10C). In the present study, the structures of CPS10B and CPS10C were determined by chemical and high resolution NMR methods to define the features of each serotype. Both CPS10C and CPS10F had β1-6-linked Galf branches formed from the termini of linear repeating units by wzy-dependent polymerization through the 4-OH of subterminal GalNAc. The only difference between these polysaccharides was the wcrC-dependent α1-2 or wcrF-dependent α1-4 linkages between Gal and ribitol-5-phosphate. The presence of one linkage or the other also distinguished the repeating units of CPS10B and CPS10A. However, whereas these polysaccharides both had β1-3-linked Galf branches linked to GalNAc, only CPS10A had additional β1-6-linked Galp branches. These Galp branches and the reaction of a CPS10A-specific monoclonal antibody were eliminated by deletion of wcrG from the cps10A locus. In contrast, deletion of this gene from the cps10B locus had no effect on the structure of CPS10B, thereby identifying wcrG as a pseudogene in this serotype. The β1-3-linked Galf branches of CPS10A and CPS10B were eliminated by deletion of wcrD from each corresponding cps locus. Deletion of this gene also eliminated wcrG-dependent β1-6-linked Galp branches from CPS10A, thereby identifying WcrG as a branching enzyme that acts on the product of WcrD. These findings provide a complete view of the molecular, structural, and antigenic features of CPS serogroup 10, as well as insight into the possible emergence of new serotypes.     

Antigenic and genetic characterization of serogroup C meningococci isolated from invasive meningococcal disease cases in Canada from 1999 to 2003

Four hundred and forty-two serogroup C Neisseria meningitidis isolates from individual invasive meningococcal disease (IMD) patients in Canada during the period 1999 to 2003 were analyzed. The majority (84%) of the serogroup C meningococci were characterized by the serotype antigen 2a and belonged to the clonal complex of electrophoretic type ET-15. However, after more than a decade of endemic disease as well as a number of outbreaks and many vaccination campaigns, both genetic and antigenic variants of the serogroup C serotype 2a meningococci were noted. Such variants include strains characterized as C:2a:P1.5 and C:2a:P1.7,1 as well as a non-serotypeable phenotype due to a mutational hot spot on the serotype 2a PorB outer-membrane protein. Meningococci characterized by the antigen formula B:2a:P1.5,2 and B:2a:P1.7,1 have also been found, which suggests capsule switching. Besides the clonal group of ET-15/ET-37, small numbers of serogroup C isolates were found to belong to the clonal complexes of ST-8 (Cluster A4), ST-41/44 (Lineage 3), ST-35, and ST-269.     

Evolution of a tRNA operon in gamma purple bacteria

Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum.     

Relapses or reinfections: Analysis of a case of Clostridium difficile-associated colitis by two typing systems

Immunoblotting and pulsed-field gel electrophoresis of Clostridium difficile isolates were employed to differentiate reinfection by a newly acquired strain from relapse by an original strain in a 10-year-old patient with four episodes of C. difficile-associated colitis. Immunoblot typing demonstrated subserogroup K-1 of serogroup K for the first and second organisms, subserogroup A-1 of serogroup A for the third organism, and subserogroup G-4 of serogroup G for the fourth organism. PFGE analysis revealed consistent results with immunoblot analysis except that the strains from the fourth episode, whose DNA constantly degraded, were nontypable by this method. Five separate isolates of C. difficile from a specimen of each episode showed identical PFGE patterns, indicating that infections of multiple strains probably did not occur in this patient. These typing results suggested that the second episode after a 17-day course of vancomycin therapy represented a relapse by the strain causing the first episode, and that the third and fourth episodes after tapering vancomycin therapy were reinfections by other strains. Both immunoblot and PFGE typing systems are promising tools for analyzing recurrence of C. difficile infection. Received: 27 November 1995 / Revised: 1 January 1996 / Accepted: 22 March 1996     

Red/ET重组及其在生物医学中的应用

通过应用Rac噬菌体的RecE RecT和λ噬菌体的RedαRedβ系统而建立的DNA工程平台———Red ET重组,是一种不依赖于限制性内切酶的分子克隆新技术。运用该技术能够介导PCR产物或寡核苷酸对目标基因进行剪切、插入、融合及突变等多种操作,在生物医学领域里具有广阔的应用前景,尤其在基因组功能研究中对BACs、PACs和细菌染色体的打靶修饰以及基因敲除动物DNA靶分子的快速构建等方面最有效。随着Red ET重组的推广与应用,该技术本身也在不断被改进,在工作效率得到显著提高的同时,其操作也变得更加简单、省时、省力。结合自身的一些研究结果,对Red ET重组的技术特点、发展现状和在生物医学中的应用进行了详细阐述。     

Genomic Relatedness of Clostridium difficile strains from different toxinotypes and serogroups

Clostridium difficile strains can be divided into sixteen toxinotypes (0 and I to XV) according to changes in their toxin genes. To determine the genomic similarity between toxinotypes, two molecular typing techniques were used, AP-PCR and PFGE. Strains were selected from five serogroups (A1, A15, E, F, X) and represented non-toxinogenic isolates, strains with toxin genes identical to the reference C. difficile strain, VPI 10463 (toxinotype 0), and strains with variant toxin genes from toxinotypes III, IV, V, VI, VII, VIII, IX, and XI. The strains studied formed three main clusters, which correlated well with serogroups: in the first were strains from serogroup A15 and E; in the second, serogroup A1 strains; and in the third, strains from serogroups F and X. Within these three clusters strains of a single toxinotype were grouped together. Toxinotypes III, IV and VIII were more similar to strains with ordinary toxin genes or non-toxinogenic isolates within the same serogroup than to other toxinotypes. Toxinotypes V, VI, VII, and XI, which exhibit similar changes in their toxin genes, seem to be more closely related one to another than to other toxinotypes. It can be concluded that variant Clostridium difficile strains do not have a common ancestor and that groups of different toxinotypes arose independently from strains with ordinary toxin genes.     

Population genetics of Vibrio vulnificus: identification of two divisions and a distinct eel-pathogenic clone

Genetic relationships among 62 Vibrio vulnificus strains of different geographical and host origins were analyzed by multilocus enzyme electrophoresis (MLEE), random amplification of polymorphic DNA (RAPD), and sequence analyses of the recA and glnA genes. Out of 15 genetic loci analyzed by MLEE, 11 were polymorphic. Cluster analysis identified 43 distinct electrophoretic types (ETs) separating the V. vulnificus population into two divisions (divisions I and II). One ET (ET 35) included all indole-negative isolates from diseased eels worldwide (biotype 2). A second ET (ET 2) marked all of the strains from Israel isolated from patients who handled St. Peter's fish (biotype 3). RAPD analysis of the 62 V. vulnificus isolates identified 26 different profiles separated into two divisions as well. In general, this subdivision was comparable (but not identical) to that observed by MLEE. Phylogenetic analysis of 543 bp of the recA gene and of 402 bp of the glnA gene also separated the V. vulnificus population into two major divisions in a manner similar to that by MLEE and RAPD. Sequence data again indicated the overall subdivision of the V. vulnificus population into different biotypes. In particular, indole-negative eel-pathogenic isolates (biotype 2) on one hand and the Israeli isolates (biotype 3) on the other tended to cluster together in both gene trees. None of the methods showed an association between distinct clones and human clinical manifestations. Furthermore, except for the Israeli strains, only minor clusters comprising geographically related isolates were observed. In conclusion, all three approaches (MLEE, RAPD, and DNA sequencing) generated comparable but not always equivalent results. The significance of the two divisions (divisions I and II) still remains to be clarified, and a reevaluation of the definition of the biotypes is also needed.     

The application of multiplex PCR to detect seven different DNA targets in group B streptococci

Group B Streptococcus (GBS) causes severe infections in infants and in immunocompromised adults. GBS pathogenicity varies between and within serotypes, with considerable variation in genetic content between strains. For this reason, it is important to be able to carry out immediate and comprehensive diagnostics of these infections. Seven genes important for screening of GBS infection were detected: cfb gene encoding the CAMP factor presented in every GBS; the cps operon genes such as cps1aH, cps1a/2/3IJ, and cps5O specific for capsular polysaccharide types Ia, III, and V, respectively; macrolide resistance genes ermB and mefA/E; and the gbs2018 S10 region specific for ST17 hypervirulent clone. Standardization of multiplex PCR with the use of seven primer pairs was performed on 81 bacterial strains representing different GBS isolates (n = 75) and other Gram-positive cocci (n = 10). Multiplex PCR can be used as an effective screening method to detect different sequences important for the screening of GBS infection.     

The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E.

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.