The last ORF of an xps gene cluster, designated xpsD, is required for the secretion of extracellular enzymes across the outer membrane in Xanthomonas campestris pv. campestris. It could encode a protein of 759 amino acid residues. A consensus N-terminal lipoprotein signal peptide was revealed from its deduced amino acid sequence. A [3H]palmitate labelling experiment indicated that XpsD was fatty-acylated. Differential extraction with Triton X-100 disclosed that XpsD was fractionated with the outer membrane. Sucrose gradient sedimentation analysis of total membranes also indicated that XpsD was mainly located in the outer membrane. At least part of XpsD is exposed to the cell surface as suggested by trypsin experiment results. Intact cells pretreated with antibody against XpsD could indirectly be labelled with fluorescent agent. When the N-terminal lipoprotein signal peptide was replaced with a nonlipoprotein signal peptide cleavable by signal peptidase I, non-fatty-acylated XpsD was synthesized. Its subcellular location was indistinguishable from that of the fatty-acylated XpsD. Complementation of an xpsD::Tn5 mutant of X. campestris pv. campestris indicated that this non-fatty-acylated XpsD remains functional in extracellular protein secretion. A stable, C-terminal truncated protein, XpsDd414-759, was synthesized from a mutated xpsD gene. Although it stayed associated with the outer membrane and exposed to the cell surface, it no longer could complement the xpsD::Tn5 mutant of X. campestris pv. campestris.

The mutant sigA allele of Bacilus subtilis DB1005 was confirmed to be temperature sensitive (ts) and transferable among strains of B. subtilis by chromosomal transformation and gene conversion. This ts sigA allele had a pleiotropic eflect on gene expression of DB1005. The induction of certain heat shock proteins in DB1005 was markedly less significant than that observed in the wild-type strain (DB2) under heat stress. In contrast, some proteins required for coping with oxidative stress and glucose starvation were induced abruptly in DB1005 but not in DB2. Heat induction of the groEL gene in vivo at both transcription and translation levels was much lower in DB1005 than in DB2. Besides, the putative cA-type promoter from the groESL operon of B. subtilis was able to be transcribed by the reconstituted or4 RNA polymerase in vitro at both 37 and 49°C. These results strongly suggest that the expression of the groEL gene of B. subtilis under heat stress is regulated at least in part by e at the level of transcription. Our results also showed that DB1005 did not respond too differently from the wild type to ethanol stress, except after a relatively long exposure.CA factor ofBacillus subtilis plays important roles both in the maintenance of vegetative growth and in the regulation of sporulation. To study the structural and functional properties as well as the roles of oA in the regulation of B. subtilis cell development, we constructed a temperature-sensitive (ts) sigA mutant named DB1005. Our data showed that the temperature sensitivity of this mutant did not result from a rapid degradation of the ts CA protein (8); other causes should be responsible for the defect. Therefore, the characterization of this mutant under heat stress becomes our main interest for the moment.It is known that a characteristic set of proteins is induced in nearly all organisms under heat shock and other forms of environmental stress (26,27,33). In Escherichia coli, the heat shock protein genes are under the control of a minor sigma factor, cr32 (18,43). The heat shock response of B. subtilis in several strains of bacilli has been studied (1, 30, 44); however, the mechanism of its regulation remains unclear. The q28 RNA polymerase was once thought to transcribe the heat shock genes of B. subtilis because of its overlapping promoter specificity with E. coli cr32 RNA polymerase (2). However, experiments which disrupted the structural gene of u28 demonstrated that this or affected only the transcription of flagellar and possibly other chemotaxis genes (20) but had nothing to do with heat shock. Recent studies indicated that the transcription starting sites of certain heat shock protein genes (groESL operon and dnaK locus) of B. subtilis were preceded by a ca'-type promoter, and no other promoter sequences recognizable by other sigma factors were identified (25,47

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The 8 factor of Bacillus subtilis DB1005 contahs two amino acid substitutions (II98A and IZOZA) in the promoter -10 binding region. It has been confirmed that this c factor is responsible for the temperature sensitivity of B. subtilis DB1005. An investigation was conducted into how the mutant aA could cause temperature-sensitive (Ts) cell growth by analysing its structural stability, cellular concentration and transcriptional activity. The mutant aA was unstable even a t the permissive temperature of 37 "C (t1,* 59 min), whereas the wildtype counterpart was fairly stable under the same conditions (tl12 > 600 min). However, neither wild-type 8 nor mutant 8 was stable a t 49 "C (tl12 34 min and 23 min, respectively). Analyses of the rates of 8 synthesis revealed that B. subtilis DB1005 was able to compensate for unstable 8 by elevating the level of aA a t 37 "C but not at 49 "C. Moreover, overexpression of the mutant 8 a t 49 "C could not suppress the Ts phenotype of B. subtilis DB1005. This indicates that the temperature sensitivity of B. subtilis DB1005 is not due to insufficient 8 concentration in the cell. The greater decline of an already reduced activity of the mutant aA a t 49 "C suggests that the temperature sensitivity of B.subtilis DB1005 is instead the result of a very low activity of 8; probably below a critical level necessary for cell growth.

Uridine triphosphate (UTP)-glucose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.9) is an enzyme that catalyzes the formation of uridine diphosphate (UDP)-glucose from UTP and glucose-1-phosphate. GalU is involved in virulence in a number of animal-pathogenic bacteria since its product, UDP-glucose, is indispensable for the biosynthesis of virulence factors such as lipopolysaccharide and exopolysaccharide. However, its function in Xanthomonas campestris pv. campestris, the phytopathogen that causes black rot in cruciferous plants, is unclear. Here, we characterized a galU mutant of X. campestris pv. campestris and showed that the X. campestris pv. campestris galU mutant resulted in a reduction in virulence on the host cabbage. We also demonstrated that galU is involved in bacterial attachment, cell motility, and polysaccharide synthesis. Furthermore, the galU mutant showed increased sensitivity to various stress conditions including copper sulfate, hydrogen peroxide, and sodium dodecyl sulfate. In addition, mutation of galU impairs the expression of the flagellin gene fliC as well as the attachment-related genes xadA, fhaC, and yapH. In conclusion, our results indicate involvement of galU in the virulence factor production and pathogenicity in X. campestris pv. campestris, and a role for galU in stress tolerance of this crucifer pathogen.

SummaryBacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant. Induction of s A has been observed exclusively in this mutant harbouring extra copies of the plasmid-borne Ts sigA gene transcriptionally controlled by the P1P2 promoters of the B. subtilis macromolecular synthesis (MMS; rpoD or sigA) operon. Investigation of the mechanisms leading to the induction has allowed us to identify a s B -type promoter, P7, in the MMS operon for the ®rst time. Therefore, at least seven promoters in total are responsible for the regulation of the B. subtilis MMS operon, including the four known s A -and s H -type promoters, as well as two incompletely de®ned promoters. The P7 promoter was activated in B. subtilis after the imposition of heat, ethanol and salt stresses, indicating that the MMS operon of B. subtilis is subjected to the control of general stress. The signi®cant heat induction of P7 in B. subtilis DB1005 harbouring a plasmid-borne Ts sigA gene can be explained by a model of competition between s A and s B for core binding; very probably, the s B factor binds more ef®ciently to core RNA polymerase under heat shock. This mechanism may provide a means for the expression of the B. subtilis MMS operon when s A becomes defective in core binding.

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BackgroundThe gram-negative Xanthomonas campestris pv. campestris is the pathogenic bacterium that causes black rot disease in crucifers. The virulence determinants of this bacterium include extracellular enzymes, exopolysaccharides, and biofilm formation. Here, one transposon mutant of X. campestris pv. campestris strain 17 that affects biofilm formation was isolated, and subsequent analyses led to the identification of the lolA gene, which encodes an outer membrane lipoprotein chaperone.ResultsThe lolA mutant exhibited significant reductions in bacterial attachment, extracellular enzyme production, virulence, and tolerance in the presence of myriad membrane-perturbing agents. These phenotypic changes of the mutant could be complemented to the wild-type level through the intact lolA gene. Proteomic analysis revealed that 109 proteins were differentially expressed after lolA mutation. These differentially expressed proteins were categorized in various functional groups and were mainly associated with the membrane component, were involved in transport, and contained receptor activity. Through reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis, deletion of lolA was determined to have caused significantly reduced expression of genes that encode the major extracellular enzymes, the biofilm-related proteins, and the virulence-related proteins. The RT-qPCR analysis also indicated that the expression of several genes that encode putative outer membrane lipoproteins and TonB-dependent receptors was reduced after lolA mutation.ConclusionsThis is the first report to define the lolA gene as a virulence factor and to contribute to the functional understanding of, and provide new information concerning, the role of lolA in Xanthomonas. Furthermore, the results of this study provide and extend new insights into the function of lolA in bacteria.Electronic supplementary materialThe online version of this article (10.1186/s12866-019-1387-9) contains supplementary material, which is available to authorized users.